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| United States Patent |
6,153,741
|
|
Richards
, et al.
|
November 28, 2000
|
DNA methylation gene from plants
Abstract
A novel gene, DDM1, and its encoded protein are provided. The gene was
isolated from a region of Arabidopsis thaliana chromosome 5. DDM1 appears
to be part of the SWI2/SNF2 family of chromatin-remodeling proteins.
Disruption of the gene results in DNA hypomethylation, among other
phenotypes. The DDM1 gene defines a novel member of the DNA methylation
system. Methods of using DDM1, and transgenic organisms comprising DDM1,
are also provided.
| Inventors:
|
Richards; Eric J. (St. Louis, MO);
Jeddeloh; Jeffrey A. (New Market, MD)
|
| Assignee:
|
Washington University (St. Louis, MO)
|
| Appl. No.:
|
104070 |
| Filed:
|
June 24, 1998 |
| Current U.S. Class: |
536/23.6; 435/69.1; 435/410; 435/411; 435/419; 536/24.1; 800/278; 800/298; 800/306 |
| Intern'l Class: |
C12N 005/04; C12N 015/29; C12N 015/82; C12N 005/00; C07H 021/04 |
| Field of Search: |
536/23.6,24.1
435/69.1,410,411,419
800/278,298,306
|
References Cited
Other References
Boase et al. In Vitro Cellular and Developmental Biology. 1998. vol. 34:
46-51.
B.R. Cairns, Trends in Biochem. 23: 20-25, 1998.
Chen & Pikaard, Genes & Devel. 11: 2124-2136, 1997.
Jeddeloh et al., Genes Devel. 12:1714-1725, 1998.
Kakutani et al., Nuc. Acids Res. 23: 130-137, 1995.
Martinez-Balbas, M.A. et al., Proc. Natl. Acad. Sci. USA 95: 132-137, 1998.
Mittlesten Scheid et al., Proc. Natl. Acad. Sci. USA 95: 632-637, 1998.
Pazin, M.J. et al., Cell 88: 737-740, 1997.
Richards, Trends in Genetics 13: 319-323, 1998.
|
Primary Examiner: Smith; Lynette R. F.
Assistant Examiner: Zaghmout; Ousama M-Faiz
Attorney, Agent or Firm: Reed; Janet E.
Saul Ewing Remick & Saul, LLP
Goverment Interests
Pursuant to 35 U.S.C. .sctn.202(c), it is acknowledged that the U.S.
Government has certain rights in the invention described herein, which was
made in part with funds from the National Science Foundation.
Parent Case Text
This application claims priority to U.S. Provisional Application Serial No.
60/083,612, filed Apr. 30, 1998, the entirety of which is incorporated by
reference herein.
Claims
What is claimed is:
1. An isolated nucleic acid molecule comprising a gene located on
Arabidopsis thaliana chromosome 5, lower arm, said gene occupying a
segment of said chromosome 5, lower arm, flanked on the centromeric side
within 20 kilobases by a gene encoding a zinc-finger protein and on the
telomeric side within 1 kilobase by a gene encoding a glutamic acid tRNA,
said gene having a restriction endonuclease cleavage map as shown in FIG.
7C.
2. The nucleic acid molecule of claim 1, wherein said gene is composed of
exons that form an open reading frame having a sequence that encodes a
polypeptide about 750-850 amino acids in length.
3. A cDNA molecule comprising the exons of the nucleic acid molecule of
claim 2.
4. The nucleic acid molecule of claim 2, wherein said open reading frame
encodes amino acid sequence I.D. No. 2.
5. The nucleic acid molecule of claim 4, which comprises an open reading
frame of Sequence I.D. No. 1.
6. A recombinant DNA molecule, comprising a vector having an insert that
includes the nucleic acid molecule of claim 1.
7. The recombinant DNA molecule of claim 6, which is cosmid C38, ATCC
Accession No. 207208.
8. An isolated nucleic acid molecule which is a gene, having a sequence
selected from the group consisting of:
a) An open reading frame defined by exons of Sequence I.D. No. 1;
(b) a sequence hybridizing with one or more exons of Sequence I.D. No. 1 or
its complement, under conditions calculated to achieve hybridization
between sequences according to a formula of: T.sub.m =81.5.degree.
C.+16.6Log[Na+]+0.41(% G+C)-0.63 (% formamide)-600/#bp in duplex;
(c) a sequence encoding a polypeptide having Sequence I.D. No. 2; and
(d) a sequence encoding a polypeptide encoded by a gene contained in cosmid
clone C38, designated ATCC Accession No. 207208.
9. A transgenic organism comprising the nucleic acid molecule of claim 1.
10. The transgenic organism of claim 9, which is a plant.
Description
FIELD OF THE INVENTION
This invention relates to the field of plant molecular biology, genetic
engineering and regulation of gene expression. In particular, this
invention provides a novel gene, DDM1, which plays an important role in
the regulation of DNA methylation, and resultant regulation of gene
expression, in plant genomic DNA.
BACKGROUND OF THE INVENTION
Plant genomes contain substantial amounts of 5-methylcytosine. Up to 20-30%
of the cytosines are methylated in the nuclear genome of many flowering
plants. As in other organisms, methylation of cytosine residues in plants
occurs post-replicatively through the action of cytosine-DNA
methyltransferases. Plant DNA methyltransferases have been characterized
biochemically, and plant genes encoding these enzymes have been isolated
by virtue of their similarity to their mammalian counterparts.
Investigations of native plant genes and transgenic plants containing
foreign genes have found a general correlation between transcriptional
inactivity and increased DNA methylation, consistent with evidence from
mammalian systems. This evidence supports a role for cytosine methylation
in maintaining transcriptional states.
The plant's need for developmental plasticity and environmental interaction
suggests that plants extensively employ epigenetic regulatory strategies.
Such strategies rely on heritable, often reversible, changes in access to
the underlying genetic information, but not alteration of the primary
nucleotide sequence. As one example, the alteration of DNA methylation is
expected to perturb plant development significantly, provided that
differential DNA methylation is an important component of epigenetic
regulation in plants.
One paradigm linking DNA methylation and developmental regulation comes
from work on the mouse, where average genome cytosine methylation levels
in embryonic lineages drop sharply in the early cleavages following
fertilization, then rise again around the time of implantation. In plants,
a similar pattern has been observed in studies of DNA methylation content
in pollen and post-embryonic tissue of varying age. Information from such
studies indicates that there is a gradual rise in 5-methylcytosine levels
in post-embryonic tissues produced by meristems at positions further from
the base of the plant (i.e., tissues of increasing age). Genetic studies
of transposon systems in maize also demonstrate an age-dependent gradient
of increasing epigenetic modification, which is correlated with DNA
methylation.
Both biochemical and genetic approaches have been taken to alter DNA
methylation in eucaryotic organisms. Methylation inhibitor treatments have
induced developmental abnormalities in many plant species. Transgenic
plants expressing antisense molecules specific for a native cytosine
methyltransferase gene have been found to exhibit genomic hypomethylation,
presumably due to the antisense interference with expression of the gene.
In another approach, mutants of Arabidopsis thaliana have been isolated,
which show a decrease in DNA methylation (ddm) resulting in reduced
nuclear 5-methylcytosine levels. The best characterized mutations define
the DDM1 gene. Homozygotes carrying recessive ddm1 alleles contain 30% of
the wild-type levels of 5-methylcytosine. The ddm1 mutations do not map to
the two known cytosine-DNA methyltransferase genes of A. thaliana, nor do
they affect DNA methyltransferase activity detectable in nuclear extracts
(Kakutani et al., Nuc. Acids Res. 23: 130-137, 1995). In addition, ddm1
mutations do not appear to affect the metabolism of the active methyl
group donor, S-adenosylmethionine (Kakutani et al., 1995, supra).
For the foregoing reasons, the DDM1 gene product is likely to be a novel
component of the DNA methylation system, or involved in determining the
cellular context (e.g., chromatin structure, subnuclear localization) of
the methylation reaction. Consequently, it would be a clear advance in the
art of plant molecular and cellular biology to identify and isolate the
DDM1 gene and/or its encoded protein. Such a gene and protein would find
utility for the purpose of modifying the methylation status of a selected
genome and thereby altering one or more regulatory features of gene
expression from that genome.
SUMMARY OF THE INVENTION
A novel gene, DDM1, and its encoded protein are provided in accordance with
the present invention. The gene has been identified as a novel element of
the DNA methylation system.
In one aspect of the invention, an isolated nucleic acid molecule
comprising a gene located on Arabidopsis thaliana chromosome 5, lower arm,
is provided. The gene occupies a segment of chromosome 5, lower arm, which
is flanked on the centromeric side within 20 kilobases by a gene encoding
a zinc-finger protein and on the telomeric side within 1 kilobase by a
gene encoding a glutamic acid tRNA. Disruption of the gene is associated
with DNA hypomethylation. The gene encodes a polypeptide of about 800
amino acids in length. The nucleotide sequence of the DDM1 gene is set
forth herein as SEQ ID NO:1 and its deduced amino acid sequence as SEQ ID
NO:2.
In another aspect of the invention, an isolated DDM1 gene is provided,
having a sequence selected from the group consisting of: (a) Sequence I.D.
No. 1; (b) an allelic variant or natural mutant of Sequence I.D. No. 1;
(c) a sequence hybridizing with part or all of Sequence I.D. No. 1 or its
complement and encoding a polypeptide substantially the same as part or
all of a polypeptide encoded by Sequence I.D. No. 1; (d) a sequence
encoding part or all of a polypeptide having amino acid Sequence I.D. No.
2; and (e) a sequence encoding part or all of a polypeptide contained in
the cosmid clone C38, designated ATCC Accession No. 207208.
According to another aspect of the invention, a polypeptide is provided,
which is produced by expression of an isolated nucleic acid molecule
comprising part or all of an open reading frame of a gene located on
Arabidopsis thaliana chromosome 5, lower arm, the gene occupying a segment
of chromosome 5, lower arm, flanked on the centromeric side within 20
kilobases by a gene encoding a zinc-finger protein and on the telomeric
side within 1 kilobase by a gene encoding a glutamic acid tRNA. This
polypeptide preferably has the amino acid sequence of part or all of
Sequence I.D. No. 2.
According to another aspect of the invention, an isolated protein encoded
by an Arabidopsis thaliana gene is provided, which is a member of an
SWI2/SNF2 family of polypeptides. Loss of function of the protein is
associated with DNA hypomethylation. The protein is encoded by a gene
located on A. thaliana chromosome 5, lower arm, centromerically flanked
within 20 kilobases by a zinc finger-encoding gene and telomerically
within one kilobase by a gene encoding a glutamic acid tRNA.
According to another aspect of the invention, a transgenic organism
comprising the DDM1 gene is provided. In one embodiment, the transgenic
organism is a plant.
In other aspects of the invention, methods are provided for stabilizing
fidelity of DNA methylation in an organism, which comprise transforming
the organism with the DDM1 gene. Methods are also provided for reducing or
eliminating gene silencing in a plant, or for inducing inbreeding
depression in a plant, which comprise inhibiting or preventing expression
of an endogenous DDM1 gene of the plant.
These aspects of the invention, as well as other features and advantages of
the invention, will be described in greater detail in the description and
examples set forth below.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Autoradiograph showing Southern analysis of F3 recombinant pools in
a ddm1 segregating family genotyping. HpaI-digested DNA from different
families (pools A-P) was electrophoresed on a 1% agarose gel, then blotted
to a nylon filter. Each pool represents about 20-30 individuals from each
family. The families were genotyped by comparing the results with the
pericentric 180 pb probe (+ladder=hypomethylated: ddm1/DDM1 or
ddm1/ddm1;-ladder=DDM1/DDM1) to the 26S rDNA probe (loss of large
fragment=ddm1/ddm1) (L=homozygous for the Landsberg allele; C=homozygous
for the Columbia allele; H=heterozygous).
FIG. 2. Diagram and summary showing scheme for positional cloning of DDM1
locus on Arabidopsis thaliana chromosome 5.
FIG. 3. Schematic diagram of thin-layer chromatography analysis of
carotenoid extracts from A. thaliana wild-type and aba mutant tissues. BC
indicates the position of beta-carotene. Chlor A & B indicates the smear
of chlorophyll A and B. The smear below leutein is Chlorophyll A and B
stripped of magnesium ions. Notice the aba plants fail to accumulate
neoxanthin and violaxanthin, and that yi has no effect on the pattern.
FIG. 4. Diagram showing the genetic and physical map of the DDM1 locus in
Arabidopsis thaliana, at lower resolution.
FIG. 5. Diagram showing the genetic and physical map of the DDM1 locus in
Arabidopsis thaliana, at higher resolution.
FIG. 6. Diagram showing the physical map of the DDM1 locus on cosmid C38 of
the A. thaliana genomic cosmid library.
FIG. 7. DDM1 gene identification. FIG. 7A: Ethidium bromide stained 2.5%
agarose gel containing size-fractionated RsaI restriction fragments of
230R-354F amplicons generated by PCR using various genomic templates
(C=Columbia, L=Landsberg, Zu=Zurich, som4 D som 8=DNA hypomethylation
mutants in a Zu background, M=molecular weight markers). A size
polymorphism is seen in the som8 sample (A vs. B) localizes the som8
mutation to the beginning of the region similar to SNF2L. FIG. 7B:
Ethidium bromide stained native polyacrylamide gel (1.times.MDE; FMC)
containing size-fractionated RsaI restriction fragments of SNF2F-SNF2R
amplicons generated by PCR using the templates indicated at the top of the
gel (c38=cosmid DNA containing the region; ddm1-x=genomic DNA from
indicated mutant). The PCR products were mixed as indicated, heat
denatured and slowly cooled following a heteroduplex analysis protocol.
The .about.700 bp RsaI fragment in the ddm1-2 mutant contains a mutation
leading to a change in electrophoretic mobility. FIG. 7C: A map of the
region indicating the position of oligonucleotide primers (230R, A, SNF2F,
354F, & SNF2R). The identity of RsaI restriction fragments from the
230R-354F amplicon are shown at the lower right. The approximate positions
of the som8 and ddm1-2 lesion are indicated above the map, and the genomic
region similar to SNF2L is shown by the shaded box beneath the map.
FIG. 8. Diagram showing DDM1 gene structure. The exon-intron structure of
the DDM1 gene is shown, as well as the positions of the two molecularly
characterized ddm1 alleles (som8 & ddm1-2).
FIG. 9. RT-PCR detection of DDM1 expression and the ddm1-2 splicing
defect--ethidium bromide stained size-fractionated products of PCR
amplification using various templates. The nature of the templates is
indicated at the top of the gel: DNA=genomic DNA from either DDM1 or
ddm1-2 plants; -RT=mock cDNA synthesized template carried out in the
absence of reverse transcriptase; +RT=first strand cDNA synthesis carried
out on polyA+RNA purified from either DDM1 or ddm1-2 plants. The predicted
size of the amplification products is shown at the bottom of the figure. K
and M' were used as oligonucleotide primers for PCR amplification. The
RT-PCR product from ddm1-2 cDNA templates shows an altered mobility
indicative of a splicing defect.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
Various terms relating to the biological molecules of the present invention
are used hereinabove and also throughout the specification and claims. The
terms "substantially the same," "percent similarity" and "percent
identity" are defined in detail below.
With reference to nucleic acids of the invention, the term "isolated
nucleic acid" is sometimes used. This term, when applied to DNA, refers to
a DNA molecule that is separated from sequences with which it is
immediately contiguous (in the 5' and 3' directions) in the naturally
occurring genome of the organism from which it was derived. For example,
the "isolated nucleic acid" may comprise a DNA molecule inserted into a
vector, such as a plasmid or virus vector, or integrated into the genomic
DNA of a procaryote or eucaryote. An "isolated nucleic acid molecule" may
also comprise a cDNA molecule.
With respect to RNA molecules of the invention the term "isolated nucleic
acid" primarily refers to an RNA molecule encoded by an isolated DNA
molecule as defined above. Alternatively, the term may refer to an RNA
molecule that has been sufficiently separated from RNA molecules with
which it would be associated in its natural state (i.e., in cells or
tissues), such that it exists in a "substantially pure" form (the term
"substantially pure" is defined below).
With respect to protein, the term "isolated protein" or "isolated and
purified protein" is sometimes used herein. This term refers primarily to
a protein produced by expression of an isolated nucleic acid molecule of
the invention. Alternatively, this term may refer to a protein which has
been sufficiently separated from other proteins with which it would
naturally be associated, so as to exist in "substantially pure" form.
The term "substantially pure" refers to a preparation comprising at least
50-60% by weight the compound of interest (e.g., nucleic acid,
oligonucleotide, protein, etc.). More preferably, the preparation
comprises at least 75% by weight, and most preferably 90-99% by weight,
the compound of interest. Purity is measured by methods appropriate for
the compound of interest (e.g. chromatographic methods, agarose or
polyacrylamide gel electrophoresis, HPLC analysis, and the like).
With respect to antibodies of the invention, the term "immunologically
specific" refers to antibodies that bind to one or more epitopes of a
protein of interest, but which do not substantially recognize and bind
other molecules in a sample containing a mixed population of antigenic
biological molecules.
With respect to oligonucleotides, the term "specifically hybridizing"
refers to the association between two single-stranded nucleotide molecules
of sufficiently complementary sequence to permit such hybridization under
pre-determined conditions generally used in the art (sometimes termed
"substantially complementary"). In particular, the term refers to
hybridization of an oligonucleotide with a substantially complementary
sequence contained within a single-stranded DNA or RNA molecule of the
invention, to the substantial exclusion of hybridization of the
oligonucleotide with single-stranded nucleic acids of non-complementary
sequence.
The term "promoter region" refers to the 5' regulatory regions of a gene.
The term "reporter gene" refers to genetic sequences which may be operably
linked to a promoter region forming a transgene, such that expression of
the reporter gene coding region is regulated by the promoter and
expression of the transgene is readily assayed.
The term "selectable marker gene" refers to a gene product that when
expressed confers a selectable phenotype, such as antibiotic resistance,
on a transformed cell or plant.
The term "operably linked" means that the regulatory sequences necessary
for expression of the coding sequence are placed in the DNA molecule in
the appropriate positions relative to the coding sequence so as to effect
expression of the coding sequence. This same definition is sometimes
applied to the arrangement of coding sequences and transcription control
elements (e.g. promoters, enhancers, and termination elements) in an
expression vector.
The term "DNA construct" refers to genetic sequence used to transform
plants and generate progeny transgenic plants. These constructs may be
administered to plants in a viral or plasmid vector. Other methods of
delivery such as Agrobacterium T-DNA mediated transformation and
transformation using the biolistic process are also contemplated to be
within the scope of the present invention. The transforming DNA may be
prepared according to standard protocols such as those set forth in
"Current Protocols in Molecular Biology", eds. Frederick M. Ausubel et
al., John Wiley & Sons, 1995.
II. Description of DDM1 and its Encoded Polyeptide
In accordance with the present invention, a novel gene, DDM1, has been
isolated from the flowering plant Arabidopsis thaliana. Through analysis
of mutant plants, this gene has been identified as important for the
maintenance of proper genomic cytosine methylation, and its function
appears to be necessary to maintain gene silencing. Biochemical and
molecular genetic results indicate that DDM1 encodes a novel component of
the DNA methylation machinery.
We have isolated the DDM1 gene from A. thaliana using a map-based cloning
approach, which is described in detail in the examples. Briefly, the DDM1
gene was initially localized to the bottom of the lower arm of chromosome
5 by reference to molecular markers segregating in an F2 family (parental
cross: Columbia ddm1/ddm1 X Landsberg erecta DDM1/DDM1). Next,
recombination breakpoints in the region surrounding a ddm1 mutation were
isolated by collecting cross-over chromosomes by reference to flanking
genetic markers. The recombination breakpoints delimited a region of
approximately 25 kilobases. Cloned DNA corresponding to this genomic
region was isolated by subcloning DNA from a bacterial artificial
chromosome (BAC) containing molecular markers mapping both proximal and
distal to the ddm1 marker. The nucleotide sequence of a single cosmid
subclone encompassing the 25 kb region was determined to identify
candidate genes. The DDM1 gene was localized to one of the seven predicted
protein-coding genes in the region by reference to DNA polymorphisms. A
well-characterized EMS-induced mutation, ddm1-2, leads to a conformational
polymorphism in a RsaI restriction fragment in the 3' end of the gene. The
ddm1-2 mutation is a base pair substitution (G.fwdarw.A) in a splice site
donor site leading to an alteration in mRNA structure (FIGS. 8 and 9).
Moreover, a putative ddm1 allele, som8, carries a structural rearrangement
at the predicted 5' end of the gene. Subsequent DNA sequence analysis
indicated that the som8 mutation is a complex rearrangement (83 bp
insertion plus a 1 bp deletion) directly following the predicted
translation start site. The som8 mutation destroys the open reading frame
of the first protein-coding exon and leads to premature termination after
only 16 codons (FIG. 8). The affected gene encodes a SWI2/SNF2-like
protein product with a large degree of amino acid similarity to yeast,
Drosophila and mammalian SWI2/SNF2-like products.
The prototype SWI2/SNF2 gene was identified in yeast as a non-lethal gene
necessary to effect expression of mating type and sucrose metabolism
genes. Subsequently, a number of related genes, some of which are
essential for growth, have been identified in yeast, Drosophila and
mammals. The proteins encoded by the SWI2/SNF2 gene family act as a part
of large, multi-subunit complexes that remodel chromatin (Pazin, M. J. et
al., Cell 88: 737-740, 1997). In most cases, these complexes act as
transcriptional activators by providing transcription factor access to the
DNA sequences packaged in the nucleosomes.
Without intending to be bound by any particular mechanism for the
functionality of the DDM1 gene product, we believe the DDM1 SWI2/SNF2-like
gene may encode a component of a remodeling complex that is specialized to
facilitate maintenance methyltransferase access to newly-replicated DNA
packaged in chromatin. This model provides an explanation for the ddm1
mutations' preferential hypomethylation of highly repeated sequences,
which are expected to be more tightly packed than single-copy sequences.
We anticipate a number of applications for our novel gene and its encoded
protein, and our discovery of the involvement of a SWI2/SNF2-like gene in
the eucaryotic DNA methylation system. Such applications are described in
greater detail below.
Although the DDM1 genomic clone from Arabidopsis thaliana is described and
exemplified herein, this invention is intended to encompass nucleic acid
sequences and proteins from other organisms, including plants, yeast,
insects and mammals, that are sufficiently similar to be used instead of
the Arabidopsis DDM1 nucleic acid and proteins for the purposes described
below. These include, but are not limited to, allelic variants and natural
mutants of Sequence I.D. No. 1, which are likely to be found in different
species of plants or varieties of Arabidopsis. Because such variants are
expected to possess certain differences in nucleotide and amino acid
sequence, this invention provides an isolated DDM1 nucleic acid molecule
having at least about 60% (preferably 70% and more preferably over 80%)
sequence homology in the coding regions with the nucleotide sequence set
forth as Sequence I.D. No. 1 (and, most preferably, specifically
comprising the coding region of sequence I.D. No. 1). This invention also
provides isolated polypeptide products of the open reading frames of
Sequence I.D. No. 1, having at least about 60% (preferably 70% or 80% or
greater) sequence homology with the amino acid sequences of Sequence I.D.
No. 2. Because of the natural sequence variation likely to exist among
DDM1 genes, one skilled in the art would expect to find up to about 30-40%
nucleotide sequence variation, while still maintaining the unique
properties of the DDM1 gene and encoded polypeptide of the present
invention. Such an expectation is due in part to the degeneracy of the
genetic code, as well as to the known evolutionary success of conservative
amino acid sequence variations, which do not appreciably alter the nature
of the encoded protein. Accordingly, such variants are considered
substantially the same as one another and are included within the scope of
the present invention.
For purposes of this invention, the term "substantially the same" refers to
nucleic acid or amino acid sequences having sequence variation that do not
materially affect the nature of the protein (i.e. its structure and/or
biological activity). With particular reference to nucleic acid sequences,
the term "substantially the same" is intended to refer to coding regions
and to conserved sequences governing expression, and refers primarily to
degenerate codons encoding the same amino acid, or alternate codons
encoding conservative substitute amino acids in the encoded polypeptide.
With reference to amino acid sequences, the term "substantially the same"
refers generally to conservative substitutions and/or variations in
regions of the polypeptide that do not affect structure or function. The
terms "percent identity" and "percent similarity" are also used herein in
comparisons among amino acid sequences. These terms are intended to be
defined as they are in the UWGCG sequence analysis program (Devereaux et
al., Nucl. Acids Res. 12: 387-397, 1984), available from the University of
Wisconsin.
The following description sets forth the general procedures involved in
practicing the present invention. To the extent that specific materials
are mentioned, it is merely for purposes of illustration and is not
intended to limit the invention. Unless otherwise specified, general
cloning procedures, such as those set forth in Sambrook et al., Molecular
Cloning, Cold Spring Harbor Laboratory (1989) (hereinafter "Sambrook et
al.") or Ausubel et al. (eds) Current Protocols in Molecular Biology, John
Wiley & Sons (1997) (hereinafter "Ausubel et al.") are used.
A. Preparation of DDM1 Nucleic Acid Molecules, Encoded Polypeptides and
Antibodies Specific for the Polypeptides
1. Nucleic Acid Molecules
DDM1 nucleic acid molecules of the invention may be prepared by two general
methods: (1) they may be synthesized from appropriate nucleotide
triphosphates, or (2) they may be isolated from biological sources. Both
methods utilize protocols well known in the art.
The availability of nucleotide sequence information, such as the cDNA
having Sequence I.D. No. 1, enables preparation of an isolated nucleic
acid molecule of the invention by oligonucleotide synthesis. Synthetic
oligonucleotides may be prepared by the phosphoramadite method employed in
the Applied Biosystems 38A DNA Synthesizer or similar devices. The
resultant construct may be purified according to methods known in the art,
such as high performance liquid chromatography (HPLC). Long,
double-stranded polynucleotides, such as a DNA molecule of the present
invention, must be synthesized in stages, due to the size limitations
inherent in current oligonucleotide synthetic methods. Thus, for example,
a long double-stranded molecule may be synthesized as several smaller
segments of appropriate complementarity. Complementary segments thus
produced may be annealed such that each segment possesses appropriate
cohesive termini for attachment of an adjacent segment. Adjacent segments
may be ligated by annealing cohesive termini in the presence of DNA ligase
to construct an entire long double-stranded molecule. A synthetic DNA
molecule so constructed may then be cloned and amplified in an appropriate
vector.
DDM1 genes also may be isolated from appropriate biological sources using
methods known in the art. In the exemplary embodiment of the invention,
the A. thaliana DDM1 clone was isolated from a BAC genomic library of A.
thaliana In alternative embodiments, cDNA clones of DDM1 may be isolated.
A preferred means for isolating DDM1 genes is PCR amplification using
genomic templates and DDM1-specific primers.
In accordance with the present invention, nucleic acids having the
appropriate level sequence homology with part or all the coding regions of
Sequence I.D. No. 1 may be identified by using hybridization and washing
conditions of appropriate stringency. For example, hybridizations may be
performed, according to the method of Sambrook et al., using a
hybridization solution comprising: 5.times.SSC, 5.times.Denhardt's
reagent, 1.0% SDS, 100 .mu.g/ml denatured, fragmented salmon sperm DNA,
0.05% sodium pyrophosphate and up to 50% formamide. Hybridization is
carried out at 37-42.degree. C. for at least six hours. Following
hybridization, filters are washed as follows: (1) 5 minutes at room
temperature in 2.times.SSC and 1% SDS; (2) 15 minutes at room temperature
in 2.times.SSC and 0.1% SDS; (3) 30 minutes-1 hour at 37.degree. C. in
2.times.SSC and 0.1% SDS; (4) 2 hours at 45-55.degree. in 2.times.SSC and
0.1% SDS, changing the solution every 30 minutes.
One common formula for calculating the stringency conditions required to
achieve hybridization between nucleic acid molecules of a specified
sequence homology (Sambrook et al., 1989):
T.sub.m =81.5.degree. C.+16.6Log [Na+]+0.41(% G+C)-0.63 (%
formamide)-600/#bp in duplex
As an illustration of the above formula, using [N+]=[0.368] and 50%
formamide, with GC content of 42% and an average probe size of 200 bases,
the T.sub.m is 57.degree. C. The T.sub.m of a DNA duplex decreases by
1-1.5.degree. C. with every 1% decrease in homology. Thus, targets with
greater than about 75% sequence identity would be observed using a
hybridization temperature of 42.degree. C. Such a sequence would be
considered substantially homologous to the sequences of the present
invention.
Nucleic acids of the present invention may be maintained as DNA in any
convenient cloning vector. In a preferred embodiment, clones are
maintained in plasmid cloning/expression vector, such as pGEM-T (Promega
Biotech, Madison, Wis.) or pBluescript (Stratagene, La Jolla, Calif.),
either of which is propagated in a suitable E. coli host cell.
DDM1 nucleic acid molecules of the invention include cDNA, genomic DNA,
RNA, and fragments thereof which may be single- or double-stranded. Thus,
this invention provides oligonucleotides (sense or antisense strands of
DNA or RNA) having sequences capable of hybridizing with at least one
sequence of a nucleic acid molecule of the present invention, such as
selected segments of the DNA having Sequence I.D. No. 1. Such
oligonucleotides are useful as probes for detecting DDM1 genes or mRNA in
test samples, e.g. by PCR amplification, or for the positive or negative
regulation of expression of DDM1 genes at or before translation of the
mRNA into proteins.
The DDM1 promoter and other expression regulatory sequences for DDM1 are
also expected to be useful in connection with the present invention.
Sequence I.D. No. 1 shows about 750 bp of sequence upstream from the
beginning of the coding region, which should contain such expression
regulatory sequences. In addition, Sequence I.D. No. 3 (see the Example)
constitutes about 5 kbp of additional upstream sequence, which may contain
other regulatory sequences, such as enhancer elements.
2. Proteins
Polypeptides encoded by DDM1 nucleic acids of the invention may be prepared
in a variety of ways, according to known methods. If produced in situ the
polypeptides may be purified from appropriate sources, e.g., plant parts.
Alternatively, the availability of nucleic acid molecules encoding the
polypeptides enables production of the proteins using in vitro expression
methods known in the art. For example, a cDNA or gene may be cloned into
an appropriate in vitro transcription vector, such a pSP64 or pSP65 for in
vitro transcription, followed by cell-free translation in a suitable
cell-free translation system, such as wheat germ or rabbit reticulocytes.
In vitro transcription and translation systems are commercially available,
e.g., from Promega Biotech, Madison, Wis. or BRL, Rockville, Md.
According to a preferred embodiment, larger quantities of DDM1-encoded
polypeptide may be produced by expression in a suitable procaryotic or
eucaryotic system. For example, part or all of a DNA molecule, such as the
coding portion of Sequence I.D. No. 1, may be inserted into a plasmid
vector adapted for expression in a bacterial cell (such as E. coli) or a
yeast cell (such as Saccharomyces cerevisiae), or into a baculovirus
vector for expression in an insect cell. Such vectors comprise the
regulatory elements necessary for expression of the DNA in the host cell,
positioned in such a manner as to permit expression of the DNA in the host
cell. Such regulatory elements required for expression include promoter
sequences, transcription initiation sequences and, optionally, enhancer
sequences.
The DDM1 polypeptide produced by gene expression in a recombinant
procaryotic or eucyarotic system may be purified according to methods
known in the art. In a preferred embodiment, a commercially available
expression/secretion system can be used, whereby the recombinant protein
is expressed and thereafter secreted from the host cell, to be easily
purified from the surrounding medium. If expression/secretion vectors are
not used, an alternative approach involves purifying the recombinant
protein by affinity separation, such as by immunological interaction with
antibodies that bind specifically to the recombinant protein. Such methods
are commonly used by skilled practitioners.
The DDM1-encoded polypeptides of the invention, prepared by the
aforementioned methods, may be analyzed according to standard procedures.
Methods for analyzing the functional activity are available. For instance,
DNA methylation levels are detectable by known methods. Alternatively, the
function of the DDM1 gene product as part of a chromatin remodeling
machine permits the use of in vitro assays for chromatin remodeling, which
are known in the art (e.g., B. R. Cairns, Trends in Biochem. 23: 20-25,
1998).
The present invention also provides antibodies capable of
immunospecifically binding to polypeptides of the invention. Polyclonal or
monoclonal antibodies directed toward the polypeptide encoded by DDM1 may
be prepared according to standard methods. Monoclonal antibodies may be
prepared according to general methods of Kohler and Milstein, following
standard protocols. In a preferred embodiment, antibodies are prepared,
which react immunospecifically with various epitopes of the DDM1-encoded
polypeptides.
B. Uses of DDM1 Nucleic Acids, Encoded Proteins and Antibodies
1. DDM1 Nucleic Acids
DDM1 nucleic acids may be used for a variety of purposes in accordance with
the present invention. The DNA, RNA, or fragments thereof may be used as
probes to detect the presence of and/or expression of DDM1 genes. Methods
in which DDM1 nucleic acids may be utilized as probes for such assays
include, but are not limited to: (1) in situ hybridization; (2) Southern
hybridization (3) northern hybridization; and (4) assorted amplification
reactions such as polymerase chain reactions (PCR).
The DDM1 nucleic acids of the invention may also be utilized as probes to
identify related genes from other species, including but not limited to,
plants, yeast, insects and mammals, including humans. As is well known in
the art and described above, hybridization stringencies may be adjusted to
allow hybridization of nucleic acid probes with complementary sequences of
varying degrees of homology. Thus, DDM1 nucleic acids may be used to
advantage to identify and characterize other genes of varying degrees of
relation to the exemplary coding sequence of Sequence I.D. No. 1, thereby
enabling further characterization of this family of genes. Additionally,
they may be used to identify genes encoding proteins that interact with
protein encoded by DDM1 (e.g., by the "interaction trap" technique).
As discussed above and in greater detail in Example 1, the similarity among
plant DDM1 and its SWI2/SNF2 counterparts in yeast, Drosophila and mammals
indicates that the functional aspects of these proteins will also be
conserved. Thus, DDM1 is expected to play an important role in DNA
methylation and resultant down-regulation of gene expression. Plants
engineered to over-express DDM1 can be expected to have improved fidelity
of the DNA methylation system. The evidence suggests that loss of DDM1
function leads to reduction in the efficiency of maintenance methylation
due to reduced accessibility of the methyltransferase enzyme to the
substrate. Hence, excess DDM1 function could lead to an increase in the
fidelity of the inheritance of DNA methylation thereby reducing the
occurrence of spurious methylation mistakes which could compromise the
organism's viability or fecundity. In fact, there are experimental data
demonstrating that loss of DDM1 function leads to stochastic
hypermethylation, and epigenetic lesion formation, as well. For these
reasons, DDM1 overexpression lines are expected to have useful properties.
Transgenic plants expressing the DDM1 gene or antisense nucleotides can be
generated using standard plant transformation methods known to those
skilled in the art. These include, but are not limited to, Agrobacterium
vectors, PEG treatment of protoplasts, biolistic DNA delivery, UV laser
microbeam, gemini virus vectors, calcium phosphate treatment of
protoplasts, electroporation of isolated protoplasts, agitation of cell
suspensions with microbeads coated with the transforming DNA, direct DNA
uptake, liposome-mediated DNA uptake, and the like. Such methods have been
published in the art. See, e.g., Methods for Plant Molecular Biology
(Weissbach & Weissbach, eds., 1988); Methods in Plant Molecular Biology
(Schuler & Zielinski, eds., 1989); Plant Molecular Biology Manual (Gelvin,
Schilperoort, Verma, eds., 1993); and Methods in Plant Molecular
Biology--A Laboratory Manual (Maliga, Klessig, Cashmore, Gruissem &
Varner, eds., 1994).
The method of transformation depends upon the plant to be transformed. The
biolistic DNA delivery method is useful for nuclear transformation. In
another embodiment of the invention, Agrobacterium vectors are used to
advantage for efficient transformation of plant nuclei.
In a preferred embodiment, the gene is introduced into plant nuclei in
Agrobacterium binary vectors. Such vectors include, but are not limited
to, BIN19 (Bevan, 1984) and derivatives thereof, the pBI vector series
(Jefferson et al., 1987), and binary vectors pGA482 and pGA492 (An, 1986).
The DDM1 gene may be placed under a powerful constitutive promoter, such as
the Cauliflower Mosaic Virus (CaMV) 35S promoter or the figwort mosaic
virus 35S promoter. Transgenic plants expressing the DDM1 gene under an
inducible promoter (either its own promoter or a heterologous promoter)
are also contemplated to be within the scope of the present invention.
Inducible plant promoters include the tetracycline repressor/operator
controlled promoter.
Using an Agrobacterium binary vector system for transformation, the DDM1
coding region, under control of a constitutive or inducible promoter as
described above, is linked to a nuclear drug resistance marker, such as
kanamycin resistance. Agrobacterium-mediated transformation of plant
nuclei is accomplished according to the following procedure:
(1) the gene is inserted into the selected Agrobacterium binary vector;
(2) transformation is accomplished by co-cultivation of plant tissue (e.g.,
leaf discs) with a suspension of recombinant Agrobacterium, followed by
incubation (e.g., two days) on growth medium in the absence of the drug
used as the selective medium (see, e.g., Horsch et al. 1985);
(3) plant tissue is then transferred onto the selective medium to identify
transformed tissue; and
(4) identified transformants are regenerated to intact plants.
It should be recognized that the amount of expression, as well as the
tissue specificity of expression of the DDM1 gene in transformed plants
can vary depending on the position of their insertion into the nuclear
genome. Such position effects are well known in the art. For this reason,
several nuclear transformants should be regenerated and tested for
expression of the transgene.
In some instances, it may be desirable to down-regulate or inhibit
expression of endogenous DDM1 in plants possessing the gene. One clear
benefit to engineering a reduction of DDM1 function is to reduce gene
(including transgene) silencing. Plant lines with reduced or absent DDM1
function are expected to be viable based on results obtained with
Arabidopsis. Further, it has been shown that gene silencing is suppressed
in ddm1 Arabidopsis lines (Jeddeloh et al., Genes Devel. 12:1714-1725,
1998). There are two other beneficial characteristics of DDM1 deficient
plant lines. First, alteration in DNA methylation leads to changes in
flowering time, and as such, is a potentially powerful tool for
manipulating plant development. (See, e.g., Richards, Trends in Genetics
13: 319-323, 1998), Second, ddm1 mutant lines exhibit inbreeding
depression (a reduction in vigor after inbreeding) (Richards, Trends in
Genetics, 1998, supra), a characteristic which may be desirable to include
in situations where proprietary germplasms in hybrid plants are at risk of
unauthorized use. For instance, a genetically engineered hybrid
(containing one or more useful transgenes) could be further engineered to
down-regulate endogenous DDM1 expression. Unauthorized inbreeding of such
lines would be discouraged because the progeny of such lines would lack
vigor.
To achieve the aforementioned benefits associated with reduced gene
expression, DDM1 nucleic acid molecules, or fragments thereof, may also be
utilized to control the production of DDM1-encoded proteins. In one
embodiment, full-length DDM1 antisense molecules or antisense
oligonucleotides, targeted to specific regions of DDM1-encoded RNA that
are critical for translation, are used. The use of antisense molecules to
decrease expression levels of a predetermined gene is known in the art. In
a preferred embodiment, antisense molecules are provided in situ by
transforming plant cells with a DNA construct which, upon transcription,
produces the antisense sequences. Such constructs can be designed to
produce full-length or partial antisense sequences.
In another embodiment, overexpression of DDM1 is induced to generate a
co-suppression effect. This excess expression serves to promote
down-regulation of both endogenous and exogenous DDM1 genes.
Optionally, transgenic plants can be created containing mutations in the
region encoding the active site of DDM1. This embodiment may be preferred
in certain instances.
From the foregoing discussion, it can be seen that DDM1 and its homologs
will be useful for introducing alterations in gene expression in an
organism, for a variety of purposes. As described above, for instance, the
Arabidopsis DDM1 gene can be used to isolate mutants or engineer organisms
that express reduced function of DDM1 orthologs. Based on results in
Arabidopsis, such mutants or engineered organisms are expected to be
viable and display valuable characteristics, such as inbreeding depression
and a reduction in gene silencing. In addition, we anticipate that
dysfunction in human DDM1 orthologs may contribute to diseases that
involve alterations in DNA methylation, including cancer (Baylin, S. B. et
al., Adv. Cancer Res. 72: 141-196, 1998) and immunodeficiency/chromosome
instability/facial anomalies syndrome (ICF) (Smeets, D. F. C. M. et al.,
Hum. Genet. 94: 240-246, 1994).
2. DDM1 Proteins and Antibodies
Purified DDM1-encoded proteins, or fragments thereof, may be used to
produce polyclonal or monoclonal antibodies which also may serve as
sensitive detection reagents for the presence and accumulation of
DDM1-encoded protein in cultured cells or tissues and in intact organisms.
Recombinant techniques enable expression of fusion proteins containing
part or all of the DDM1-encoded protein. The full length protein or
fragments of the protein may be used to advantage to generate an array of
monoclonal or polyclonal antibodies specific for various epitopes of the
protein, thereby providing even greater sensitivity for detection of the
protein in cells or tissue.
DDM1 gene products also may be useful as pharmaceutical agents if it is
determined that DDM1 loss of function plays a role in carcinogenesis, as
mentioned above. The gene products could be administered as replacement
therapy for persons having neoplasias associated with DDM1 loss of
function.
Polyclonal or monoclonal antibodies immunologically specific for
DDM1-encoded proteins may be used in a variety of assays designed to
detect and quantitate the protein. Such assays include, but are not
limited to: (1) flow cytometric analysis; (2) immunochemical localization
in cultured cells or tissues; and (3) immunoblot analysis (e.g., dot blot,
Western blot) of extracts from various cells and tissues.
Polyclonal or monoclonal antibodies that immunospecifically interact with
the polypeptide encoded by DDM1 can be utilized for identifying and
purifying such proteins. For example, antibodies may be utilized for
affinity separation of proteins with which they immunospecifically
interact. Antibodies may also be used to immunoprecipitate proteins from a
sample containing a mixture of proteins and other biological molecules.
The following specific example is provided to illustrate embodiments of the
invention. It is not intended to limit the scope of the invention in any
way.
EXAMPLE
Molecular Isolation of the DDM1 Locus From Arabidopsis thaliana
DDM1 is likely to be a novel component of the DNA methylation machinery
because the known components had been eliminated by genetic and
biochemical analysis. We decided that positionally isolating the DDM1
locus would provide insight as to the mode of ddm1-induced
hypomethylation. Crude mapping experiments had positioned the ddm1 locus
on the south arm of A. thaliana chromosome five. Based upon our map
position and the relative genetic distance to some known genetic markers
for which there existed a genotype in our segregating families, we
believed that the ddm1 lesions should lie between the mutations known as
yi (yellow inflorescence) and aba (abscisic acid deficient) (see FIG. 2).
Generation of recombinant chromosomes would be necessary for the
positional cloning strategy.
COLLECTION OF RECOMBINATION BREAKPOINTS AROUND DDM1:
Our strategy is outlined in FIG. 2. Our ddm1 mutations were isolated in the
Columbia strain background and back-crossed to this parent six times, and
then selfed to generate a segregating ddm1 family. We chose the Landsberg
strain to cross against allowing us to follow parental origin of any
region of the genome. The Landsberg yi aba line was kindly provided by Dr.
Jan Zeevart, Michigan State University.
We first crossed a YI ddm1 ABAI YI DDM1 ABA line in the Columbia background
to a Landsberg yi DDM1 aba/yi DDM1 aba line. One half of the F1 would be
the desired genotype YI ddm1 ABA/yi DDM1 aba. These F1 were identified by
progeny testing-scoring for plants which segregated ddm1 hypomethylated
DNA. These ddm1/DDM1 F1 plants contain hypomethylated pericentric DNA in
their silique tissue that can be detected in these pools by Southern
analysis, as in the original screen for isolating the mutants.
Selfed seed from F1 YI ddm1 ABA/yi DDM aba were collected dried and then
sown. Individuals that possess a recombination breakpoint in the yi aba
interval were recovered by screening the F2 progeny for the following
visible phenotypes: yi ABA and YI aba. yi plants are detected .sup..about.
1 wk post germination by the presence of a yellow inflorescence. aba
mutant individuals were identified by two methods. First, a wilting test
was done by shifting the plants from the 85% relative humidity in the
growth chamber to the dry lab air. aba plants cannot correctly control the
turgor pressure of their stomatal guard cells and rapidly wilt. This
screen was prone to mis-identifying aba mutants so a secondary screen was
also used. The secondary verification of the aba mutant phenotype was done
by thin layer chromatography (TLC) (FIG. 3). The phytohormone abscisic
acid (ABA) is produced as a final byproduct of carotenoid biosynthesis.
aba mutants are blocked before aba can be produced. As a result they fail
to produce two carotenoids, neoxanthin and violaxanthin. aba mutants lack
two blue bands just above the origin (corresponding to the carotenoids
neoxanthin and violaxanthin). 135 F2 plants were recovered that were
either yi ABA or YI aba. The predicted genetic distance between yi and aba
was determined to be .sup..about. 8 cM (integrated map 5/92), which meant
that recombinants were expected to be recovered at about a frequency of
.sup..about. 10%.
It was critical to use a ddm1/+ plant rather than a ddm1/ddm1 plant in this
cross because ddm1 hypomethylated DNA is not remethylated in a wt
background (Vongs et al., science 260: 1926-1928, 1993). Because the
hypomethylated chromosomes are not remethylated in a DDM1 nucleus,
hypomethylated chromosome segments will each segregate as a Mendelian
locus. Since we use the methylation status of the rDNA and centromere to
score the genotype at ddm1, segregation of hypomethylated chromosome
segments in addition to the ddm1 mutation could result in the
mis-genotyping of a potential recombination event. The entire problem was
avoided by using a heterozygous parent as the donor for the ddm1 mutation,
because the mutation is fully recessive, no hypomethylated DNA was
introduced into the cross and no miss-genotyping could occur later.
DNA was prepared from 111 of the 135 recombinant lines by growing F3
progeny tissue. The F3 DNA samples were scored for the DNA hypomethylation
phenotype diagnostic of ddm1. HpaII digests were done and two Southern
blots were performed. First, the centromeric (180 bp) repeat probe was
used to identify lines which were either heterozygous or homozygous for
ddm1 in the F2 generation. Second, we used a 26S rDNA probe (FIG. 1). Only
F2 plants homozygous for the ddm1 mutation would show complete
hypomethylation with the 26S probe. Therefore lines homozygous for ddm1
should show a ladder with the centromeric probe and complete
hypomethylation with the rDNA probe. Lines heterozygous for ddm1 show
hypomethylation only with the centromeric probe, and lines wild-type for
DDM1 show no ladders at all. In the 111 lines, 84 recombination events
occurred between YI and ddm1 and 36 events occurred between ddm1 and ABA.
This placed ddm1 in the YI ABA interval and closer to ABA than YI.
Once all of the lines were scored for ddm1, the lines were scored for two
available molecular markers predicted to be in the interval, CATHHANK and
BIO205. Of the 87 lines scored for all three internal markers 17 lines
were recombinant between CATHHANK and ddm1, and 9 lines were recombinant
between ddm1 and BIO205. The interval between CATHHANK and BIO205 was
predicted to be 2.6 cM (Recombinant Inbred map 6/95
(http://nasc.nott.ac.uk)). With 28 recombination events in 2.6 cM we had 1
crossover per 0.09 cM. In Arabidopsis the average number of Kb per cM is
200, suggesting that we had an average of one crossover every 19 Kb
through the interval. At this stage we stopped collecting recombinants and
focused on refining the interval by placing new and recently developed
markers onto the genetic map. At the same time we utilized the available
flanking molecular markers (CATHHANK and BIO205) to begin to assemble a
physical map of the region (Table 1 and FIG. 4).
TABLE 1
______________________________________
Genetic Markers for Positioning DDM1
Marker Type Enzyme Provided by
______________________________________
LFY CAPS RsaI Research Genetics
pCITd77 RFLP BsaAI A.th. Stock Ctr
CATHHANK SSLP G. Picard
AB5-13 RFLP not tested
H. Goodman
g4510 RFLP DraI A.th. Stock Ctr
cT10D21(i)2-12H3
RFLP XbaI developed
cT10D21(i)2-5.7H3
RFLP XbaI developed
Gap5 CAPS RsaI developed
105N23T7 RFLP BclI/ClaI A. th Stock Ctr
cT10D21(i)50
RFLP PstI developed
sT10D21Bam RFLP BclI developed
sT8D13NcoI RFLP BclI/BsaAI
developed
Mi335 RFLP HindIII C. Dean
93N23T7 RFLP XbaI A.th Stock Ctr
m555R1.4 RFLP DraI H. Adler
m555 CAPS AccI/MseI J. Bender
Bio205 RFLP DraI B. Osborne
BIO205-17 RFLP DraI developed
CD3-42 RFLP BglII A.th Stock Ctr
______________________________________
The table indicates the molecular markers used in the positional cloning
of DDM1., their nature (RFLP = restriction fragment length polymorphism,
SSLP = simple sequence length polymorphism, CAPS = cleaved amplified
polymorphic sequence), the enzyme necessary to detect the polymorphism,
and the provider of the clone. A.th Stock Ctr = Arabidopsis Stock Center,
Ohio State University.
BUILDING A PHYSICAL MAP OF THE DDM1 LOCUS:
A subclone of BIO205 was generated (205-17), which contained only the DNA
that looked across the polymorphic DraI restriction site. Because CATHHANK
was a simple sequence length polymorphism (SSLP), it would not be useful
as a hybridization probe. However, the marker CATHHANK comes from an
ankryin repeat clone AB5-13. Four Arabidopsis yeast artificial chromosome
(YAC) libraries were screened using AB5-13 and 205-17 as hybridization
probes. The CIC library, EG, EW, and yUP libraries were provided on
filters in 96 well formats by the A.th Stock Center (Ohio State), and were
screened simultaneously with both probes. A collection of YACs was
identified and further characterized (FIG. 4). Extensive characterization
of most YAC clones revealed that either the inserts were deleted,
chimeric, or too large to be of use. We abandoned YACs for BACs (bacterial
artificial chromosomes), which are easier to use and may possess more
insert stability.
During the course of the walk, an EST RFLP marker for Succinate
dehydrogenase (105N23T7) was discovered to have linkage to the bottom arm
of chromosome five. We subsequently showed the marker to be very tightly
linked to ddm1. Because 105N23T7 showed no recombination with ddm1 in our
breakpoint lines we reasoned that the gene must be in close proximity to
SuDH. Armed with a probe at molecular zero (the physical window of no
recombination with ddm1) we abandoned YACs using BAC filter sets provided
by Joe Ecker's group at the University of Pennsylvania, we isolated
bacterial artificial chromosomes (BACs) that contained 105N23T7 by
hybridization to the gridded filters in a 384 well format. Once we had
collected the positive clones (.about.30 in total) we used Southern
analysis to assay the BACs for marker content and assembled them into a
contig (Table 2 and FIG. 5).
TABLE 2
__________________________________________________________________________
YAC and BAC content (by hybridization)
Marker:
CATHHANK
g4510 105N23
sT10D21Bam
sT8013Nco
Mi335
93N13
M555
Bio205
CD342
__________________________________________________________________________
Clone
yCIC3B1G
+ + + + + + + + + +
yCIC5B13P
+ +
yCIC7A12 +
yEG8E5
+
yEW12E6 + + + +
yUP17F6 + +
yUP4G7 + + + + +
bF10010 + + +
bF16H19 + + +
bF19G16 + + +
bF23A16 + + +
bF26B24 + +
bF28L19 + +
bF3L3 + +
bF4L12 + + +
bF7I18 + + + +
bF9H17 + +
BT8D13 + +
bT10D21 + + +
bT13E12 + + +
bT29C4
+ +
__________________________________________________________________________
YAC clones, indicated by yXXX, and BAC clones, indicated by bXXX were
assayed for content by Southern analysis with the markers indicated acros
the top of the table.
The clones came from two libraries, TAMU (Texas A&M University), and IGF
(Germany -Max Planck Institute). We knew that 105N23T7 was at molecular
zero and that .DELTA.G4510 (a deletion derivative of the original clone,
G4510) and Mi335 were markers that flanked 105N23T7 on the centromere and
telomere sides respectively (FIG. 5). From the contig we chose two BACs,
T10D21 and T8D13 and made end clones for each by plasmid rescue. Each of
the end clones was then used as an RFLP probe and mapped on the breakpoint
lines to define the ends of the clones genetically. The results of this
mapping suggested that DDM1 was entirely on T10D21. The BAC end clone
sT10D21Bam mapped two recombination events to the right of ddm1. Because
T10D21 contained .DELTA.G4510, 105N23T7, and sT10D21Bam we had narrowed
the gene to an approximately 110 Kb interval with 10 recombination events
on the BAC. T10D21's insert was checked for chimerism and integrity by
genomic Southern using six different restriction enzymes. No alterations
of the insert relative to genomic DNA were found.
T10D21 was then amplified and a cosmid library was generated from the BAC
using Stratagene's SuperCos vector, by cloning partially digested Sau3A1
BAC insert DNA into the BamHI site of SuperCos. Ligated products were
packaged using a size selecting packaging extract (Gigapack XL
-Stratagene) and infected into E.coli XL1 MRA (Stratagene). Clones were
inoculated into a microtiter dish and then replicated using a "hedgehog"
onto a nylon filter. The colonies were screened by hybridization using
105N23T7 as a probe until about 50 positives were in hand. The positives
were then cut with HindIII and BglII to fingerprint them for contig
assembly and subsequently screened by Southern using the 105N23T7 probe.
Two overlapping cosmid clones, C38 and C50, were selected for further
analysis. Indirect end-labeling was used to generate a partial HindIII and
complete PstI restriction map of both cloned inserts (FIG. 5).
To further delimit the position of the DDM1 gene, restriction fragments
from C38 and C50 were used to find RFLP markers for genotypic analysis on
the recombination breakpoint collection. An RFLP recognized by a 5.7 Kb
HindIII fragment contained totally within C38 near it's left most
boundary, mapped +4 recombination events to the left (centromeric) of
ddm1. Further, C38 also contained a PstI polymorphism originally found
using the whole C50 cosmid as a probe, c50PstI (Table 1). c50PstI mapped 2
recombination events to the right (telomeric ) of ddm1. By restriction
mapping we were able to show that this RFLP was within 4 Kb of the
smallest PstI fragment inside SuDH. Based upon these data, we knew that
C38 contained the DDM1 gene. In the physical interval defined by C38,
.about.45 Kb, the physical distance per recombination in our break points
lines was 1 crossover every 7.5 Kb (45 Kb/6 breaks).
As the genetic mapping was proceeding, we began to look for cDNA clones
from the region. A .lambda.-PRL2 A. thaliana cDNA library (Arabidopsis
stock center Ohio State) was screened for cDNAs using isolated restriction
fragments from C38 (partial map FIG. 5). Four SuDH clones and one unknown
cDNA, provisionally named T1, were identified by using the .about.10 Kb
PstI I fragment. The representation of SuDH clones in this library was 7
SuDH-hybridizing clones per 10,000 plaques screened. PstI fragment B (8.5
Kb) recognized an unknown zinc finger protein, provisionally named Z2.
PstI fragment E (5.0 Kb) only identified more SuDH clones. Ti was
positioned distal to SuDH and as such was excluded as a DDM1 candidate. Z2
was placed centromeric to SuDH but was located .about.10 Kb away, a
distance suggesting that it may not be ddm1. We were unable to generate a
molecular marker for Z2 and as such were not able to map it relative to
ddm1, and could not exclude it as a candidate gene. Because of the high
expression levels and representation of SuDH in the cDNA library, and
because development of new markers was difficult, we concluded the best
approach to identify DDM1 was to sequence the entire C38 cosmid. The
entire sequence would afford us all of the genes and the ability to
generate molecular markers that would help further delimit the DDM1 gene.
SEQUENCING OF THE DDM1 LOCUS:
C38 DNA was amplified, purified and verified by restriction analysis. The
DNA was sonicated and 1-2 Kb fragments were isolated and purified from an
excised agarose gel slice. The isolated fragments were cloned into the
SmaI site of M13mp18. Ligated products were electroporated into E. coli
JS5 and plated on LB with Xgal and IPTG (for screening out clones without
inserts). Positive plaques were toothpicked into E. coli JM101 and a
library archive was created by making glycerol stocks in microtiter
dishes. 645 clones were amplified and phage DNA was recovered. Sequencing
was done first using the M13-21 forward primer. After assembly of the
"forward" reads, clones on the 5' and 3' ends of each sequence contig were
selected for "reverse" reads. Phage from the archived library from these
reads were amplified and RF DNA was isolated using the Wizard spin-column
miniprep kit (Promega). "Reverse" reads were sequenced from 72 double
stranded RF DNA clones with the M13mp18-45 reverse primer. Sequence data
were collected using an automated sequencer (both an ABI 373 and ABI 377)
with the accompanying software suites for the Macintosh. Following
assembly of the reverse reads, and the forward reads, six sequence contigs
were formed representing almost 50 of 52 Kb (insert .about.45Kb plus the
cosmid vector .about.7 Kb). The data set included 598 total reads from 789
total reactions (.about.75% success). Oligonucleotide primers were ordered
to close the areas corresponding to the gaps by PCR. The order of the
sequence contigs was assigned by blastx, available restriction mapping and
Southern blot analysis of C38, and confirmed by PCR. The sequence gaps
were closed by sequencing the PCR products corresponding to the gaps. The
hole sizes were 700 bp, 600 bp, 300 bp, and 3 holes were less than 200 bp.
Sequence assembly was performed using XBAP, PHRED, and PHRAP for UNIX
systems, and DNASTAR (V3.0) for the Macintosh.
BLASTx of both NCBI/GenBank and AtDB, Genefinder and NetPlantGene searches
determined that there were 10 ORFs on C38 (FIG. 6). Of these ten, at least
9 were represented in the EST library collection of Atdb, including SuDH,
Z2 and T1. The organization of the genes and predicted products are (from
centromere to telomere): AP2-like homeobox transcription factor, two
unknown ORFs, ATN1 kinase like, Z2- Zn finger, SNF2-like (SNF2L), Glu
tRNA, SuDH, Scarecrow/RGA like transcription factor, Unknown ORF,
Fibrillin like gene. The genetic interval containing ddm1 contained 5 ORFs
(from +4 L to +2 R FIG. 6). However, given that 4 recombination events
existed centromeric to SuDH (itself at 0), and that only 4 Kb telomeric to
SuDH was +2 R, we felt that by position the SNF2-like gene and Z2 were the
best candidates for ddm1. Further positioning ddm1 came by finding of a
RsaI CAPS in the GAP5 region (FIGS. 7, panel A). GAP5 is the .about.1.7 kb
that fell between the Z2 sequence contig and the SNF2L sequence contig.
Scoring of the polymorphism on the recombination breakpoints revealed that
GAP5 was at molecular zero as well. Therefore, the most proximal
breakpoint in our collection must fall to the left of SNF2L.
IDENTIFICATION OF A DDM1 CANDIDATE GENE:
Nine candidate ddm1 alleles exist, three from our EMS collection, and 6
putative alleles identified in a screen for loss of gene silencing
(ddm1=som; Mittlesten Scheid et al., Proc. Natl. Acad. Sci. USA 95:
632-637, 1998). Five of these alleles came from a fast-neutron
mutagenesis, and as such were likely to contain structural rearrangements
at the ddm1 locus.
Using the GAP5 primers, one of the FN candidate alleles (som8) was found to
contain a .about.100 bp duplication/insertion in a .about.1.3 Kb RsaI
fragment predicted to contain the ATG of the SNF2-like gene (by BCM
Genefinder) (FIG. 7, panel A). Another mutation was detected in a RsaI
digest of ddm1-2 PCR amplified DNA. The entire putative SNF2-like coding
region was amplified by PCR generating a .about.4 Kb fragment (primers A
to SNF2R) (FIG. 7, panel B). The alteration was detected following
restriction of the PCR product with RsaI using a 6% nondenaturing Mutation
Detecting Enhancing (MDE.TM.) poly-acrylamide gel (FMC). Restriction
fragments were visualized by ethidium bromide fluorescence. MDE.TM. gels
sieve not only by size but also by shape (secondary and tertiary
structures) (FMC). Analysis revealed that ddm1-2 showed an alteration in a
.about.700 bp product from the 3' end of the SNF2-like ORF. The alteration
is not evident on 2.5% or 3.5% agarose gels, suggesting that the change is
not a simple duplication/insertion making the fragment larger.
Our initial mutation detection results were confirmed by subsequent
sequence analysis (FIGS. 8 and 9). The som8 mutation results in an
insertion of 82 base pairs directly downstream of the initiator ATG codon.
The som8 mutation destroys the open reading frame of the first
protein-coding exon and leads to premature termination after only 16
codons (FIG. 8). The ddm1-2 mutation is a base pair substitution (GD>A) in
the splice donor site for intron 9. As shown in FIG. 9, the ddm1-2
mutations leads to an alteration in mRNA structure, most likely choice of
an alternative splice donor site upstream. Both the som8 and ddm1-2
mutations are expected to destroy, or severely impair, gene function.
These results identify the DDM1 gene and provide evidence that the gene is
expressed.
The nucleotide sequence of the DDM1 gene from Arabidopsis thaliana is set
forth below as Sequence I.D. No. 1 (SEQ ID NO:1). The DDM1 protein coding
regions are at the following positions: 782-1252, 1354-1440, 1549-1895,
1976-2165, 2251-2437, 2559-2629, 2703-2892, 2975-3070, 3148-3242,
3317-3436, 3540-3659, 3745-3843, 3934-4038, 4130-4354). The tRNA-glu
coding region is at positions 4826-4755. Positions 785 and 3243 are bold
and underlined to indicate: (1) the som8 rearrangement, which comprises a
deletion of G at 785 and an 83 bp insertion between 785 and 786 in the
wild-type sequence; and (2) the ddm1-2 base pair substitution of G to A at
position 3243.
TGATCATTTT CTTCCTCCGG CCAATTTGCA GATCGAAAAA TGATTTAGCT TTTTATTAAA
AATATTGTTA TTCGTTTTTA GCCGATATCA TAACTTTTTG AGATACATTA TCAACACACT
CGTGCAACTG AGATATTCTT GACACAATTT TTGCATTTGA AATTGGCAAT TTTGTACTAC
TCATATAGTT TGAAGCTTCA ATTCACTACA AAGGTTATTA CTAATTGTGT CGACAAATCC
AGCAGATTTA ATAATGCCCA TTCCATTAAA TGTTTTTTAG TTTAATAATA GGATGATCAT
ATGACCAAAA TCGTAAATAA GGGTTAGGGG TAAACCTGTC ATTTCAAGCT TCCCGCCCAT
GGGCGCTACT CCCAATTTAA TAAAAAATAA GAAAATAGGC GTAAATATGA GAGTGTGTTT
TTTCAATATA CCCTCGGTTT TGAATTTGCT CTCAAAAGCG ACGGAGACGA CTGTTTGGCT
CGGTGATTTC TCCCGCCGTT TGGGTTTTTC TTACCGGAAT TTCCTTCTCC TTCGATGGTT
AGTCTGCGCT CCAGAAAAGT TATTCCGTAA GTCCCTCCAC CTTTCCTTTT CATTTCGTTA
TTTCCGGCGA TTTTCTAGGT CCTTAACGCT CTCGAAATCG CTCGCTGTTC TTGGTGGTTT
TTGGTTCCCT CTCTGCGTAA TTTTGTTTGT CGTGTTTTTG GATTATATTC TCTGACTATT
GGTCTCACTG TTGATTTATC ATTTCTCGAT TTTGGATTTT TGGACTCTTA GGGCTTCGGA
AATGGTCAGC GACGGGAAAA CGGAGAAAGA TGCGTCTGGT GATTCACCCA CTTCTGTTCT
CAACGAAGAG GTTTGTTCTA TGTTCTACTA TTTTGCCTTC CTAGTGTGGT TGCTTTGTGA
AACTTTGTGT GTTACTCTTT GTTTCTTTAA ATCTGGGGTG TTCTGTAAAT GGGTCCTTTT
TGGTCCTTTT TTTCTGAATG TGAAGGAAAA CTGTGAGGAG AAAAGTGTTA CTGTTGTAGA
GGAAGAGATA CTTCTAGCCA AAAATGGAGA TTCTTCTCTT ATTTCTGAAG CCATGGCTCA
GGAGGAAGAG CAGCTGCTCA AACTTCGGGA AGATGAAGAG AAAGCTAACA ATGCTGGATC
TGCTGTTGCT CCTAATCTGA ATGAAACTCA GTTTACTAAA CTTGATGAGC TCTTGACGCA
AACTCAGCTC TACTCTGAGT TTCTCCTTGA GAAAATGGAG GATATCACAA TTGTAATCTT
CTTTATTTCT TTCTTCTTTG TGGTTTCTCA CTTTTCGAAT GGGAGTCATT ATTCTTAGTT
TGAACAACTT GTGGGTGAAA TTTGTTTTGC TAGAATGGGA TAGAAAGTGA GAGCCAAAAA
GCTGAGCCCG AGAAGACTGG TCGTGGACGC AAAAGAAAGG CTGCTTCTCA GTACAACAAT
GTTGGTTCCA TTTATATAAT TTTCAACTAC TATGCATGAT CTTGTATATA TTGTTTTTTC
TGCTTGTTTG AGAAAGTAAC TTACTTGGAT GCTTTTTTCT TCAATCAGAC TAAGGCTAAG
AGAGCGGTTG CTGCTATGAT TTCAAGATCT AAAGAAGATG GTGAGACCAT CAACTCAGAT
CTGACAGAGG AAGAAACAGT CATCAAACTG CAGAATGAAC TTTGTCCTCT TCTCACTGGT
GGACAGTTAA AGTCTTATCA GCTTAAAGGT GTCAAATGGC TAATATCATT GTGGCAGAAT
GGTTTGAATG GAATATTAGC TGATCAAATG GGACTTGGAA AGACGATTCA AACGATCGGT
TTCTTATCAC ATCTGAAAGG GAATGGGTTG GATGGTCCAT ATCTAGTCAT TGCTCCACTG
TCTACACTTT CAAATTGGTT CAATGAGATT GCTAGGTACT CTCATGGCCA TATGTGTTTG
TATAGATCCA ATGCTTTGGG GTTTCTGTTG AAAGTTTTCT TACCTTTTCC ATTAGGTTCA
CGCCTTCCAT CAATGCAATC ATCTACCATG GGGATAAAAA TCAAAGGGAT GAGCTCAGGA
GGAAGCACAT GCCTAAAACT GTTGGTCCCA AGTTCCCTAT AGTTATTACT TCTTATGAGG
TTGCCATGAA TGATGCTAAA AGAATTCTGC GGCACTATCC ATGGAAATAT GTTGTGATTG
ATGAGGTAAA TTCCGAGATT GGTCAATGTA CTAGGCTTTG AAGATCAAGA TGATCTCTCT
AACTGATAAT TTTGTTCTTG TATATTATAG GGCCACAGGT TGAAAAACCA CAAGTGTAAA
TTGTTGAGGG AACTAAAACA CTTGAAGATG GATAACAAAC TTCTGCTGAC AGGAACACCT
CTGCAAAATA ATCTTTCTGA GCTTTGGTCT TTGTTAAATT TTATTCTGCC TGACATCTTT
ACATCACATG ATGAATTTGA ATCATGGTAC AAACATGGTC CTTTTCTACT ATTATCCCTA
ACTAGTCTTC TTTTTTTTTT TTTTTTTGTT AACACTGGTG GCAGCTTTTT GACATTTATT
CCTTTCTTAG TATCTAACTG ATAGATGAGT CTCTACAGGT TTGATTTTTC TGAAAAGAAC
AAAAACGAAG CAACCAAGGA AGAAGAAGAG AAAAGAAGAG CTCAAGTATG TACAATTATA
TCAATTTTCC TTTATTTCTT TGATTGTATT TATGTCTTAT GCTAAGGGTA CATCTTGTCT
AGGTTGTTTC CAAACTTCAT GGTATACTAC GACCATTCAT CCTTCGAAGA ATGAAATGTG
ATGTTGAGCT CTCACTTCCA CGGAAAAAGG AGATTATAAT GTATGCTACA ATGACTGATC
ATCAGAAAAA GTTCCAGGAA CATCTGGTGA ATAACACGTT GGAAGCACAT CTTGGAGAGA
ATGCCATCCG AGGTACATGA TCTATTTTTT TTTTTTAATA CTTTGTTTAA TTATGTCATT
TTCTGCATTG ATTTGTTCAT CCCCTATACT TCAGGTCAAG GCTGGAAGGG AAAGCTTAAC
AACCTGGTCA TTCAACTTCG AAAGAACTGC AACCATCCTG ACCTTCTCCA GGGGCAAATA
GATGGTTCAT GTATGTCAGT TTCTTTTAAG AAACGTAAGA AAAACTTCTG TCATACTGTT
CTGTCTAATT GTTTCATTTC GTGACAGATC TCTACCCTCC TGTTGAAGAG ATTGTTGGAC
AGTGTGGTAA ATTCCGCTTA TTGGAGAGAT TACTTGTTCG GTTATTTGCC AATAATCACA
AAGTATGTTT CACAAACCCA TGGCTCGTAG CTCATTTCCC TTTGAGAACT TCTCTGATCC
ATTTGCTGAT GACCAGGTCC TTATCTTCTC CCAATGGACG AAACTTTTGG ACATTATGGA
TTACTACTTC AGTGAGAAGG GGTTTGAGGT TTGCAGAATC GATGGCAGTG TGAAGCTGGA
TGAAAGGAGA AGACAGGTTT CACCTGTGCT TATGCTGCTT TTGCGTTGCT TTTAAGCAAT
ATTCTGACCA AATATTATAA CCATAAGGTC TCTCTCTCTC TCTCTTTGCC TTGAAACAGA
TTAAAGATTT CAGTGATGAG AAGAGCAGCT GTAGTATATT TCTCCTGAGT ACCAGAGCTG
GAGGACTCGG AATCAATCTT ACTGCTGCTG ATACATGCAT CCTCTATGAC AGCGACTGGG
TAATCAAATC AATTAATTTA TTTTCTTTGA AGGAAAATCT TTCTCTTTCG TGTTGTCTCC
AACTGTGTTT TGTCTGATCT CCAGAACCCT CAAATGGACT TGCAAGCCAT GGACAGATGC
CACAGAATCG GGCAGACGAA ACCTGTTCAT GTTTATAGGC TTTCCACGGC TCAGTCGATA
GAGGTAAAAC TCTTTGTTGT TCATATCAAT CAATCTTAAC TTCAAACCAT TGAGATTGTT
GCCTCATGAG ATTGGTTTAT GACATTTGCT CAGACCCGGG TTCTGAAACG AGCGTACAGT
AAGCTCAAGC TGGAACATGT GGTTATTGGC CAAGGGCAGT TTCATCAAGA ACGTGCCAAG
TCTTCAACAC CTTTAGAGGT TTTAACTTCT CTTAAAGCTC AATCCTTTTT AGATACACTT
ATTATCAACA AAATCTCCTA TTGACAGCTT GAACCAAACT AACACACAGG AAGAGGACATA
CTGGCGTTGC TTAAGGAAG ATGAAACTGC TGAAGATAAG TTGATACAAA CCGATATAAG
CGATGCGGAT CTTGACAGGT TACTTGACCG GAGTGACCTG ACAATTACTG CACCGGGAGA
GACACAAGCT GCTGAAGCTT TTCCAGTGAA GGGTCCAGGT TGGGAAGTGG TCCTGCCTAG
TTCGGGAGGA ATGCTGTCTT CCCTGAACAG TTAGGACACA TTAATAAGCC AGGCCTTGAA
ACCACTTCTG TGTTTTTTTT TTTTTTTTCC GGAACATGAT CGGTTACTTT TGGCTGGGAG
GATTTAATTA TTAGAGGGCT CGGAAGTTTT TGTAAGTTAA AGAACTCACT TAAAACCCTG
AAAACATGAC AGTTAATGGT GATTAGCTCT CAATGTGATG AAAACAATTG GCCCTCTGAT
TTTGCTGTTG CGGTAATATT ATGACTTGTG TACGTTTATA GTCTTTGTAG TCTGCAATTT
TGGCATTGAG CTATTTCTCA CGAACTTATG GGATCTTATG TTTTGGATTT GGGATTTGTT
AACTTATATG ATTAGGCTCA ATAGTTTCAC AGAATATTAA AAACTTGAGT AGGGTTTAAA
AAAGAAGCAA AAAGCTCCGA TGCCGGGAAT CGAACCCGGG TCTCCTGGGT GAAAGCCAGA
TATCCTAACC GCTGGACGAC ATCGGATTTG TTGATGTCTA TTCTTGTAAA TAGTAAATAT
TTAGTTTTAT CGGTTTTGCA TCTAATGGAC TAAAACATGA ACACGAGACG CCGACAAGAA
TGAATGGGGC AGGCACCAAA CATTTGGGTA AAAGTATGCA GTGGGGTATT ATTGACAATT
TGACCATTAC AAGAGCTAAT
The deduced amino acid sequence encoded by Sequence I.D. No. 1 is set forth
below as Sequence I.D. No. 2 (SEQ ID NO:2).
MVSDGKTEKD ASGDSPTSVL NEEVCSMFYY FAFVVWLLCE TLCVTLCFFK SGVFCKWVLF
GPFFLNVKEN CEEKSVTVVE EEILLAKNGD SSLISEAMAQ EEEQLLKLRE DEEKANNAGS
AVAPNLNETQ FTKLDELLTQ TQLYSEFLLE KMEDITINGI ESESQKAEPE KTGRGRKRKA
ASQYNNTKAK RAVAAMISRS KEDGETINSD LTEEETVIKL QNELCPLLTG GQLKSYQLKG
VKWLISLWQN GLNGILADQM GLGKTIQTIG FLSHLKGNGL DGPYLVIAPL STLSNWFNEI
ARFTPSINAI IYHGDKNQRD ELRRKHMPKT VGPKFPIVIT SYEVAMNDAK RILRHYPWKY
VVIDEGHRLK NHKCKLLREL KHLKMDNKLL LTGTPLQNNL SELWSLLNFI LPDIFTSEDE
FESWYKHGLI FLKRTKTKQP RKKKRKEELK YVVSKLHGIL RPFILRRMKC DVELSLPRKK
EIIMYATMTD HQKKFQEHLV NNTLEAHLGE NAIRGQGWKG KLNNLVIQLR KNCNHPDLLQ
GQIDGSYLYP PVEEIVGQCG KFRLLERLLV RLFANNHKQV LIFSQWTKLL DIMDYYFSEK
GFEVCRIDGS VKLDERRRQI KDFSDEKSSC SIFLLSTRAG GLGINLTAAD TCILYDSDWN
PQMDLQAMDR CHRIGQTKPV HVYRLSTAQS IETRVLKRAY SKLKLEEVVI GQGQFEQERA
KSSTPLEEED ILALLKEDET AEDKLIQTDI SDADLDRLLD RSDLTITAPG ETQAAEAFPV
KGPGWEVVLP SSGGMLSSLN S
The approximately 5 kilobases of nucleotide sequence upstream of DDM1 is
set forth below as Sequence I.D. No. 3 (SEQ ID NO:3).
TGTCGGTTTCCATGGAAGATTGTGACCACGACGATGAAGCTGAAGATTCTGGTCACGTTGAAACCTTTGTTACAG
AT
TTCGCAAACGAATCGATTCGTTGCCATAAGTGTTTTAGGTGACAAAGCTATCACTTCAGCGTCTGGATCTGAAT
TTAGAC
AATCAGTGAGAACAACTAAAAACAGAAAATTTCAAACTCAAAAAACAGAAAAAAAAAAGTTTGGATTTTTGAGA
AGTACC
AGGCATTCCAGGAAGATTCCGTTTCTTCTTCCCGACGGATTTAGGAGTTAGATTTTGGTTTCCGGTCGATGAGA
CGCTTG
CATCGCCGGAAACTGTAGAGGAATTATCTAAATCAACCGGCATGTTTCAAAGATACTAAATTCCAATCTTTGAA
CACAAA
AAGGAAGAAGCAAATCTCAGCTCAGCTCAATCTAGGGTTTATCATCCTCCTCCTACTCTGTTTAGTCTCTCTTT
CTCTCT
CTCTTCTTCAGCTACCAGTCAATCTGCTTTTCGTAAAAATCTCCTTTTCCCCTTTCCGCCACCAAACTTTTCTG
ATAACT
CACTCTCTGACCTCTCTTCTTCAAAAAGATTTAAAACCCCCAAAAGAAAAAGAAAAAAAATCAAAACTTCATTA
CCCAAG
AAATCTCTTAATCATTTAACCCAGACTCTTTCTTCTCCACACGCATCTTTTATCCACCGTCCACCGATCTGATC
CAACGG
CTGAGATTTCACCGGAGACGAGTTATCCTTACTACTTCCGGCTTGTTTCTCTCTGAAGAATCACCGGAAAAAAA
AATAAG
GCGGCTTGTGTGTGAGACTTTGTGTGAAAGCTTCAACCTTTTTTTTCTTTTTCTTTGGCTTGTCCAAGAAAAAG
GAGCCT
TCTTCTTCTTTTCTCTCTCTGGAGACAATTATACTAATTTTTTTCTTTTCAACTTTTCACCCTTTTTTTTTTGT
TAACAA
ACATTTTTTATACATAATTGTGTCGACTTTCAAGTTCCAAGTATCTAAATCTGTATTTTGGACTCCCATGCAAA
TAATTA
AAATAGAATAATCTTTTTGTAGATTTTAAATTGAAAACGGTGTAGAAAGGTTAAAAGCACCAAACAAAACGAGT
AAATAG
ATATTGTAATAATTTTTTCACCTTTATGGAAAAGATTATATCATAGACGATGTACACAGATGAAAATTAGAAAA
TGGCAT
GTGAATATATGCAGTACCCATTGAATGCAATATCAGGTTTGTATTATTTTTCTATTGTATCTCTACATGTTACG
TAATCA
AACGATCAAGTAATTTATTAATATTGTCGATGGCGTAGAAATTATAAATTTATTTTATGTCATTGTTTACTATA
TAGATT
TTGAGCTAAACGACTTATTTTGTCAAAAGATATATCCGTGTTTGGTTTAAGATTGGGTTTTAGTATTTCCAATA
TTAATC
TAAATTCTTAGCTTATGAACATGTCAATAAACAAAAAAATTATTTTACTGTCACTGTCCTTAGACGGGGACAAA
GGAGGG
TATTACCGTCGCGTTGTCGGACCGTAAAATAATTAACCAAATTTTGTTGTTGAACGAATAACATTTTTTACTGT
GGGAAT
TTGTCGTGTAGCATTACGTTCGAAATCGCAATTTGTTTTCTTCTTTGTGGGTGTATATTTCTGGTTAACGAAAC
TATAAC
CCAATTTAATGCAATGTTCGTCTGTTTTTGTTGACTTTGACCCTTTTTTGGTAATATTCGTTCAGCTTTTGTTT
TAACGT
TTTCATTGCCTTGTAGGCATCTGAGAAGCTCAGATTCTGACACGTGTCTTTTGTTATCTGAATTTGCATCCGTT
GGATAA
ACATGACGCTGACAGGTGGATTGAAAAGTAACCAGCTTGGATTTCTGTGTATATGTTACACCGCCACTTCCCTT
AATTTC
TTCGTTCTTAGTTAAAATAAAAAAGGTTTATTTATGAGTAAAAGTATGTAAAACGACAACGAATTACTATAAGA
ATTAAA
ATTTATCTTTGCTTAGTAATTTGCACTTAAGATTGGATTCAAATTTTGTAAAAAGCGAATGTTACATATATGTC
CATTGA
AAAAATTGCATTTGACTTTACAAGCATTGAAATTAATTAATTTGGGACCCCTTTTTTTGTTAGTTTCAAAGGAA
GAATTA
TTTTAGGCTGAGATGGGTCCCTCCATAAACTCACTATTCTGCCAGCATACAAATTCCTTAACATATGGTCCAAA
TAGCAG
TTCCAACCACTAGTATCCAATAATAATCTGAACAAATTATCTTTCTTTTTTTTTCCTGATAATCTTGTATTTGT
TTGTTC
AATGAGCTTAATACGTATATTAGTTATGACTTATAACTAAATACTTTGACTCACTTGATCCGTACACATTGATT
TCGTTT
ATTCAAATCCGAACAACGTAATGATCTTTTTGGGCCGAGTTATTTGTATTCTCAACCTGAGTCCAACCATGCTT
TATGGG
CTTTTCTGTTTATTTATGCATGTAAAGTTTATAATGCTTGCAAATAACCACATATTGTATGAATGTAATTACTA
TGATTT
AAGGGCACTGCTTTTCTGTTTTCACGTTGTTTTCGAAATTGCTATTGCGTGTGATATCTGTGTTGGACCAATTA
TTGAAA
AGGACAAGGCTGACTCTGGTTTTTAATGAGTAGTCCCCATGGGAGTTATGTTCATTTACCACACATTTTTTTGT
ATAGTA
TAGTATGAGTTTTTATTTGATATCTTTTATCTTCGGAAAATAAATGGTTCAAATTGTTTGTCTAAAAATGCACA
CATGAA
TATCTTGTGGTCTCACACAATTGTAGGAAACAAATTAATATTTGTTGCGAAAATAATGTTATTATTTTATCATA
CGAAAT
CCTAGAGAAAATGGTGGCAAAAGAGGCAAAGACTAAACTAATGAATTTAAAATATGAAAATGATGGAATGACTG
GTTTAC
CAATATTACAGTATATTGTAATTTTATAAAAACGAATCCTGAAGAAGAGGGCAAACCCCAAGACCACGCAAATC
AGTCTA
CAAATATGAAAATTTCCAATAACTAGAAAAACATGTGCATTTATCTTTTTCCATCATTCGGATTTTTACAATGG
AAATTT
TGACCACTGAGCGCAAGTGTTATAGTATTTTATTATTATCCAATATTAATATCATTATTCGGATCCATGCATTC
TATATA
ACTATGTCCACCATCTTACTTGTGTCTATGTTGCAACTTCAACGTCGTATATATATAGGGATTGTTGTCACGAA
TACAAT
GCTAATTAAGGAAGATTGTGACTTCTCGGAAAATTTAGAACTAATTAAGAGTGGAACTAAAATGCCAATGAAAA
TAGCCT
AAATCAAAGGAGAACCACAAATATAAATTGGAAGACCTTAAAAAACAATTAAACGAGGACGAAACAAATTTTGG
AATCAT
CAATTATACGAAAAAAAGAAGAAAGAAAAAAGAGGTTTCATGAATCACAGTAGTGCTGACAATCTTCGAACCAT
TTGTGG
GTTTCATACAATCGATCACCAATAGAACAAAAGAGAAACAGAGGAACAGAAAGAATAGAAGGAGTGGGAAGTGT
ATGAGG
AAGCTGTGTCCGAACATAGACAAAGACGATGGTCTGGAGACGGTGTTGGAAGTTCCGATACCGGAGGAGATGTT
TTCCGG
TATGGGCAACAACGTTGCACTTAGGTGGCAAAATATGATGACGTGGATGAAAGCTCAAACGTCTGATAAATGGT
CGCAAC
CGCTTATCGCCGCTCGTATCAACGAGCTCCGGTTCCTTCTCTACCTCGTTGGCTCGCCTCTTATACCTCTCCAG
GTTCAA
GTCGGTCACTCTGTTCATAAGCCCGTCAAAGATTGCTCCATTGTAAGTCATTCAAAATCAATCCTTATGAAAAC
ATAACA
AAGATGTTGAAAATATGATTCCTCTTTTTTTTTTCTTTTTTTCTTTTATGATCAAAACCCAAAAAAGTCATTAC
CCTGCT
TCGTAAGTATTCAACATAAAGTTGTTAATCCATGTGTTGTACTCTGCAAGTCTGCATTACATTATTCATCGTAC
ACAGAG
TCATCAACTTCAGTTTCATTGTTTTTTTGCTTATGAATTACGATTGCAGCAAGCTTCAACGGCGAAATACATTG
TACAGC
AGTACATAGCAGCGACGGGAGGACCACAGGCGTTAAACGCCGTGAACAGCATGTGCGTCACGGGACAAGTGAAG
ATGACG
GCGTCGGAGTTTCATCAAGGAGATGATTCGGGCGTTAATCTAAAGAGCAACGACGAAATGGGTGGTTTCGTTTT
ATGGCA
AAAGGATCCAGATCTTTGGTGTTTGGAGCTCGTCGTCTCCGGTTGCAAAGTGGATATGTGGAAGCAACGGTCGG
CTTTCA
TGGCGACATTCCTCTAACCAGCAAACTCCGGCGTCTACSGGAACGCCAARACCTCTCCGCCGGTTTWTACAGGT
CCAATC
CGGTTATTGATTTTTTTTTKGATGTAATGTCCGGTTCTCAAAATGTTGAACCGGTGGTTTATTTATTGTTTGGA
GCAGGG
GTTARATCCTCGTTCGACGGCGAATCTGTTTCTTGACGCAAACGTGTATCGGAGAGAAGATAATCAACGGTGAG
GATTGC
TTTATCTTGAAACTGGAGACGAGTCCGGCGGTTCGAGAAGCTCAAAGCGGTCCGAATTTTGAGATAATTCATCA
CACGAT
ATGGGGTTATTTTAGTCAAAGATCGGGACTTTTGATTCAGTTCGAAGATTCGCGGCTTTTGAGAATGAGGACCA
AGGAAG
ACGAAGATGTCTTCTGGGAGACTAGTGCTGAGTCGGTGATGGATGATTACCGATACGTTGACAATGTGAACATC
GCTCAC
GGCGGGAAAACATCGGTCACGGTTTTCCGGTACGGTGAAGCGTCGGCGAATCATCGGAGACAGATGACGGAGAA
GTGGAG GATAGAAGAAGTTGATTTTAATGTTTGGGGTCTCTCCGTT
SNF2-like genes encode DNA-dependent ATP-hydrolyzing enzymes (with seven
characteristic "helicase" domains) that function as part of chromatin
remodeling complexes (Cairns et al., 1998, supra). Chromatin remodeling is
the ATP dependent repositioning of nucleosomes on DNA. Generally,
SNF2-like proteins act to give other proteins, such as transcription
factors, access to DNA. A number of chromatin remodeling complexes have
been characterized in yeast, Drosophila and mammals which have different
subunit composition and diverse functional roles. Arabidopsis has many
members of the SNF2-like gene family represented in the sequenced EST
collection (J. A. Jeddeloh (unpublished data)). DDM1 is not in the EST
database, suggesting that it is in fact a new member of the Arabidopsis
SNF2-like gene family.
MODELS FOR THE ACTION OF DDM1 AS A SNF2-LIKE GENE:
Models for the action of the DDM1 gene product as a SNF2-like gene fall
into two classes. The most parsimonious model is the "direct interaction"
model. Under the "direct interaction" model DDM1 acts to give DNMTase
access to cytosines, perhaps by pushing nucleosomes out of the way of the
methyltransferase. In this scenario, mutations in DDM1 would lead to
hypomethylation directly, and the phenotypic effects of ddm1 mutations
would result from methylation loss. Alternatively, DDM1 may act to
establish chromatin identity, (for example, by facilitating
heterochromatin assembly) which subsequently dictates the interaction with
the DNA methylation system. In this model, ddm1 mutations would lead to a
loss of chromatin identity, creating an unrecognizable/unmethylatable
template for the DNMTase. Under the "chromatin identity" model, ddm1
mutations compromise a determinate of chromatin structure, such as histone
acetylation state, and the loss of methylation is a secondary consequence
of the primary change in chromatin identity. A SNF2-like gene function
(ISWI) is believed to associate with histone deacetylase (Martinez-Balbas,
M. A. et al., Proc. Natl. Acad. Sci. USA 95: 132-137, 1998). If DNA
methylation and histone deacetylation lie in the same pathway, as
suggested by the findings of Chen and Pikaard (Chen & Pikaard, Genes &
Devel. 11: 2124-2136, 1997), then perhaps DDM1 mediates the interaction
between the two processes.
While certain of the preferred embodiments of the present invention have
been described and specifically exemplified above, it is not intended that
the invention be limited to such embodiments. Various modifications may be
made thereto without departing from the scope and spirit of the present
invention, as set forth in the following claims.
__________________________________________________________________________
# SEQUENCE LISTING
- <160> NUMBER OF SEQ ID NOS: 3
- <210> SEQ ID NO 1
<211> LENGTH: 5000
<212> TYPE: DNA
<213> ORGANISM: Arabidopsis thaliana
<220> FEATURE:
<221> NAME/KEY: CDS
<222> LOCATION: (782)...(1252)
<221> NAME/KEY: CDS
<222> LOCATION: (1354)...(1440)
<221> NAME/KEY: CDS
<222> LOCATION: (1549)...(1895)
<221> NAME/KEY: CDS
<222> LOCATION: (1976)...(2165)
<221> NAME/KEY: CDS
<222> LOCATION: (2251)...(2437)
<221> NAME/KEY: CDS
<222> LOCATION: (2559)...(2629)
<221> NAME/KEY: CDS
<222> LOCATION: (2703)...(2892)
<221> NAME/KEY: CDS
<222> LOCATION: (2975)...(3070)
<221> NAME/KEY: CDS
<222> LOCATION: (3148)...(3242)
<221> NAME/KEY: CDS
<222> LOCATION: (3317)...(3436)
<221> NAME/KEY: CDS
<222> LOCATION: (3540)...(3659)
<221> NAME/KEY: CDS
<222> LOCATION: (3745)...(3843)
<221> NAME/KEY: CDS
<222> LOCATION: (3934)...(4038)
<221> NAME/KEY: CDS
<222> LOCATION: (4130)...(4354)
<221> NAME/KEY: CDS
<222> LOCATION: (4826)...(4755)
<223> OTHER INFORMATION: t-RNA-glu coding region
<220> FEATURE:
<221> NAME/KEY: mutation
<222> LOCATION: (785)...(786)
<223> OTHER INFORMATION: som8 rearrangement, deletion - # of G at 785
and
#and 786sertion of 83 bp between 785
<220> FEATURE:
<221> NAME/KEY: mutation
<222> LOCATION: (3243)...(3243)
<223> OTHER INFORMATION: ddm1-2 base pair substitu - #tion of G to A
- <400> SEQUENCE: 1
- tgatcatttt cttcctccgg ccaatttgca gatcgaaaaa tgatttagct tt - #ttattaaa
60
- aatattgtta ttcgttttta gccgatatca taactttttg agatacatta tc - #aacacact
120
- cgtgcaactg agatattctt gacacaattt ttgcatttga aattggcaat tt - #tgtactac
180
- tcatatagtt tgaagcttca attcactaca aaggttatta ctaattgtgt cg - #acaaatcc
240
- agcagattta ataatgccca ttccattaaa tgttttttag tttaataata gg - #atgatcat
300
- atgaccaaaa tcgtaaataa gggttagggg taaacctgtc atttcaagct tc - #ccgcccat
360
- gggcgctact cccaatttaa taaaaaataa gaaaataggc gtaaatatga ga - #gtgtgttt
420
- tttcaatata ccctcggttt tgaatttgct ctcaaaagcg acggagacga ct - #gtttggct
480
- cggtgatttc tcccgccgtt tgggtttttc ttaccggaat ttccttctcc tt - #cgatggtt
540
- agtctgcgct ccagaaaagt tattccgtaa gtccctccac ctttcctttt ca - #tttcgtta
600
- tttccggcga ttttctaggt ccttaacgct ctcgaaatcg ctcgctgttc tt - #ggtggttt
660
- ttggttccct ctctgcgtaa ttttgtttgt cgtgtttttg gattatattc tc - #tgactatt
720
- ggtctcactg ttgatttatc atttctcgat tttggatttt tggactctta gg - #gcttcgga
780
- aatggtcagc gacgggaaaa cggagaaaga tgcgtctggt gattcaccca ct - #tctgttct
840
- caacgaagag gtttgttcta tgttctacta ttttgccttc gtagtgtggt tg - #ctttgtga
900
- aactttgtgt gttactcttt gtttctttaa atctggggtg ttctgtaaat gg - #gtcctttt
960
- tggtcctttt tttctgaatg tgaaggaaaa ctgtgaggag aaaagtgtta ct - #gttgtaga
1020
- ggaagagata cttctagcca aaaatggaga ttcttctctt atttctgaag cc - #atggctca
1080
- ggaggaagag cagctgctca aacttcggga agatgaagag aaagctaaca at - #gctggatc
1140
- tgctgttgct cctaatctga atgaaactca gtttactaaa cttgatgagc tc - #ttgacgca
1200
- aactcagctc tactctgagt ttctccttga gaaaatggag gatatcacaa tt - #gtaatctt
1260
- ctttatttct ttcttctttg tggtttctca cttttcgaat gggagtcatt at - #tcttagtt
1320
- tgaacaactt gtgggtgaaa tttgttttgc tagaatggga tagaaagtga ga - #gccaaaaa
1380
- gctgagcccg agaagactgg tcgtggacgc aaaagaaagg ctgcttctca gt - #acaacaat
1440
- gttggttcca tttatataat tttcaactac tatgcatgat cttgtatata tt - #gttttttc
1500
- tgcttgtttg agaaagtaac ttacttggat gcttttttct tcaatcagac ta - #aggctaag
1560
- agagcggttg ctgctatgat ttcaagatct aaagaagatg gtgagaccat ca - #actcagat
1620
- ctgacagagg aagaaacagt catcaaactg cagaatgaac tttgtcctct tc - #tcactggt
1680
- ggacagttaa agtcttatca gcttaaaggt gtcaaatggc taatatcatt gt - #ggcagaat
1740
- ggtttgaatg gaatattagc tgatcaaatg ggacttggaa agacgattca aa - #cgatcggt
1800
- ttcttatcac atctgaaagg gaatgggttg gatggtccat atctagtcat tg - #ctccactg
1860
- tctacacttt caaattggtt caatgagatt gctaggtact ctcatggcca ta - #tgtgtttg
1920
- tatagatcca atgctttggg gtttctgttg aaagttttct taccttttcc at - #taggttca
1980
- cgccttccat caatgcaatc atctaccatg gggataaaaa tcaaagggat ga - #gctcagga
2040
- ggaagcacat gcctaaaact gttggtccca agttccctat agttattact tc - #ttatgagg
2100
- ttgccatgaa tgatgctaaa agaattctgc ggcactatcc atggaaatat gt - #tgtgattg
2160
- atgaggtaaa ttccgagatt ggtcaatgta ctaggctttg aagatcaaga tg - #atctctct
2220
- aactgataat tttgttcttg tatattatag ggccacaggt tgaaaaacca ca - #agtgtaaa
2280
- ttgttgaggg aactaaaaca cttgaagatg gataacaaac ttctgctgac ag - #gaacacct
2340
- ctgcaaaata atctttctga gctttggtct ttgttaaatt ttattctgcc tg - #acatcttt
2400
- acatcacatg atgaatttga atcatggtac aaacatggtc cttttctact at - #tatcccta
2460
- actagtcttc tttttttttt tttttttgtt aacactggtg gcagcttttt ga - #catttatt
2520
- cctttcttag tatctaactg atagatgagt ctctacaggt ttgatttttc tg - #aaaagaac
2580
- aaaaacgaag caaccaagga agaagaagag aaaagaagag ctcaagtatg ta - #caattata
2640
- tcaattttcc tttatttctt tgattgtatt tatgtcttat gctaagggta ca - #tcttgtct
2700
- aggttgtttc caaacttcat ggtatactac gaccattcat ccttcgaaga at - #gaaatgtg
2760
- atgttgagct ctcacttcca cggaaaaagg agattataat gtatgctaca at - #gactgatc
2820
- atcagaaaaa gttccaggaa catctggtga ataacacgtt ggaagcacat ct - #tggagaga
2880
- atgccatccg aggtacatga tctatttttt ttttttaata ctttgtttaa tt - #atgtcatt
2940
- ttctgcattg atttgttcat cccctatact tcaggtcaag gctggaaggg aa - #agcttaac
3000
- aacctggtca ttcaacttcg aaagaactgc aaccatcctg accttctcca gg - #ggcaaata
3060
- gatggttcat gtatgtcagt ttcttttaag aaacgtaaga aaaacttctg tc - #atactgtt
3120
- ctgtctaatt gtttcatttc gtgacagatc tctaccctcc tgttgaagag at - #tgttggac
3180
- agtgtggtaa attccgctta ttggagagat tacttgttcg gttatttgcc aa - #taatcaca
3240
- aagtatgttt cacaaaccca tggctcgtag ctcatttccc tttgagaact tc - #tctgatcc
3300
- atttgctgat gaccaggtcc ttatcttctc ccaatggacg aaacttttgg ac - #attatgga
3360
- ttactacttc agtgagaagg ggtttgaggt ttgcagaatc gatggcagtg tg - #aagctgga
3420
- tgaaaggaga agacaggttt cacctgtgct tatgctgctt ttgcgttgct tt - #taagcaat
3480
- attctgacca aatattataa ccataaggtc tctctctctc tctctttgcc tt - #gaaacaga
3540
- ttaaagattt cagtgatgag aagagcagct gtagtatatt tctcctgagt ac - #cagagctg
3600
- gaggactcgg aatcaatctt actgctgctg atacatgcat cctctatgac ag - #cgactggg
3660
- taatcaaatc aattaattta ttttctttga aggaaaatct ttctctttcg tg - #ttgtctcc
3720
- aactgtgttt tgtctgatct ccagaaccct caaatggact tgcaagccat gg - #acagatgc
3780
- cacagaatcg ggcagacgaa acctgttcat gtttataggc tttccacggc tc - #agtcgata
3840
- gaggtaaaac tctttgttgt tcatatcaat caatcttaac ttcaaaccat tg - #agattgtt
3900
- gcctcatgag attggtttat gacatttgct cagacccggg ttctgaaacg ag - #cgtacagt
3960
- aagctcaagc tggaacatgt ggttattggc caagggcagt ttcatcaaga ac - #gtgccaag
4020
- tcttcaacac ctttagaggt tttaacttct cttaaagctc aatccttttt ag - #atacactt
4080
- attatcaaca aaatctccta ttgacagctt gaaccaaact aacacacagg aa - #gaggacat
4140
- actggcgttg cttaaggaag atgaaactgc tgaagataag ttgatacaaa cc - #gatataag
4200
- cgatgcggat cttgacaggt tacttgaccg gagtgacctg acaattactg ca - #ccgggaga
4260
- gacacaagct gctgaagctt ttccagtgaa gggtccaggt tgggaagtgg tc - #ctgcctag
4320
- ttcgggagga atgctgtctt ccctgaacag ttaggacaca ttaataagcc ag - #gccttgaa
4380
- accacttctg tgtttttttt ttttttttcc ggaacatgat cggttacttt tg - #gctgggag
4440
- gatttaatta ttagagggct cggaagtttt tgtaagttaa agaactcact ta - #aaaccctg
4500
- aaaacatgac agttaatggt gattagctct caatgtgatg aaaacaattg gc - #cctctgat
4560
- tttgctgttg cggtaatatt atgacttgtg tacgtttata gtctttgtag tc - #tgcaattt
4620
- tggcattgag ctatttctca cgaacttatg ggatcttatg ttttggattt gg - #gatttgtt
4680
- aacttatatg attaggctca atagtttcac agaatattaa aaacttgagt ag - #ggtttaaa
4740
- aaagaagcaa aaagctccga tgccgggaat cgaacccggg tctcctgggt ga - #aagccaga
4800
- tatcctaacc gctggacgac atcggatttg ttgatgtcta ttcttgtaaa ta - #gtaaatat
4860
- ttagttttat cggttttgca tctaatggac taaaacatga acacgagacg cc - #gacaagaa
4920
- tgaatggggc aggcaccaaa catttgggta aaagtatgca gtggggtatt at - #tgacaatt
4980
# 500 - #0
- <210> SEQ ID NO 2
<211> LENGTH: 801
<212> TYPE: PRT
<213> ORGANISM: Arabidopsis thaliana
- <400> SEQUENCE: 2
- Met Val Ser Asp Gly Lys Thr Glu Lys Asp Al - #a Ser Gly Asp Ser Pro
# 15
- Thr Ser Val Leu Asn Glu Glu Val Cys Ser Me - #t Phe Tyr Tyr Phe Ala
# 30
- Phe Val Val Trp Leu Leu Cys Glu Thr Leu Cy - #s Val Thr Leu Cys Phe
# 45
- Phe Lys Ser Gly Val Phe Cys Lys Trp Val Le - #u Phe Gly Pro Phe Phe
# 60
- Leu Asn Val Lys Glu Asn Cys Glu Glu Lys Se - #r Val Thr Val Val Glu
#80
- Glu Glu Ile Leu Leu Ala Lys Asn Gly Asp Se - #r Ser Leu Ile Ser Glu
# 95
- Ala Met Ala Gln Glu Glu Glu Gln Leu Leu Ly - #s Leu Arg Glu Asp Glu
# 110
- Glu Lys Ala Asn Asn Ala Gly Ser Ala Val Al - #a Pro Asn Leu Asn Glu
# 125
- Thr Gln Phe Thr Lys Leu Asp Glu Leu Leu Th - #r Gln Thr Gln Leu Tyr
# 140
- Ser Glu Phe Leu Leu Glu Lys Met Glu Asp Il - #e Thr Ile Asn Gly Ile
145 1 - #50 1 - #55 1 -
#60
- Glu Ser Glu Ser Gln Lys Ala Glu Pro Glu Ly - #s Thr Gly Arg Gly Arg
# 175
- Lys Arg Lys Ala Ala Ser Gln Tyr Asn Asn Th - #r Lys Ala Lys Arg Ala
# 190
- Val Ala Ala Met Ile Ser Arg Ser Lys Glu As - #p Gly Glu Thr Ile Asn
# 205
- Ser Asp Leu Thr Glu Glu Glu Thr Val Ile Ly - #s Leu Gln Asn Glu Leu
# 220
- Cys Pro Leu Leu Thr Gly Gly Gln Leu Lys Se - #r Tyr Gln Leu Lys Gly
225 2 - #30 2 - #35 2 -
#40
- Val Lys Trp Leu Ile Ser Leu Trp Gln Asn Gl - #y Leu Asn Gly Ile Leu
# 255
- Ala Asp Gln Met Gly Leu Gly Lys Thr Ile Gl - #n Thr Ile Gly Phe Leu
# 270
- Ser His Leu Lys Gly Asn Gly Leu Asp Gly Pr - #o Tyr Leu Val Ile Ala
# 285
- Pro Leu Ser Thr Leu Ser Asn Trp Phe Asn Gl - #u Ile Ala Arg Phe Thr
# 300
- Pro Ser Ile Asn Ala Ile Ile Tyr His Gly As - #p Lys Asn Gln Arg Asp
305 3 - #10 3 - #15 3 -
#20
- Glu Leu Arg Arg Lys His Met Pro Lys Thr Va - #l Gly Pro Lys Phe Pro
# 335
- Ile Val Ile Thr Ser Tyr Glu Val Ala Met As - #n Asp Ala Lys Arg Ile
# 350
- Leu Arg His Tyr Pro Trp Lys Tyr Val Val Il - #e Asp Glu Gly His Arg
# 365
- Leu Lys Asn His Lys Cys Lys Leu Leu Arg Gl - #u Leu Lys His Leu Lys
# 380
- Met Asp Asn Lys Leu Leu Leu Thr Gly Thr Pr - #o Leu Gln Asn Asn Leu
385 3 - #90 3 - #95 4 -
#00
- Ser Glu Leu Trp Ser Leu Leu Asn Phe Ile Le - #u Pro Asp Ile Phe Thr
# 415
- Ser His Asp Glu Phe Glu Ser Trp Tyr Lys Hi - #s Gly Leu Ile Phe Leu
# 430
- Lys Arg Thr Lys Thr Lys Gln Pro Arg Lys Ly - #s Lys Arg Lys Glu Glu
# 445
- Leu Lys Tyr Val Val Ser Lys Leu His Gly Il - #e Leu Arg Pro Phe Ile
# 460
- Leu Arg Arg Met Lys Cys Asp Val Glu Leu Se - #r Leu Pro Arg Lys Lys
465 4 - #70 4 - #75 4 -
#80
- Glu Ile Ile Met Tyr Ala Thr Met Thr Asp Hi - #s Gln Lys Lys Phe Gln
# 495
- Glu His Leu Val Asn Asn Thr Leu Glu Ala Hi - #s Leu Gly Glu Asn Ala
# 510
- Ile Arg Gly Gln Gly Trp Lys Gly Lys Leu As - #n Asn Leu Val Ile Gln
# 525
- Leu Arg Lys Asn Cys Asn His Pro Asp Leu Le - #u Gln Gly Gln Ile Asp
# 540
- Gly Ser Tyr Leu Tyr Pro Pro Val Glu Glu Il - #e Val Gly Gln Cys Gly
545 5 - #50 5 - #55 5 -
#60
- Lys Phe Arg Leu Leu Glu Arg Leu Leu Val Ar - #g Leu Phe Ala Asn Asn
# 575
- His Lys Gln Val Leu Ile Phe Ser Gln Trp Th - #r Lys Leu Leu Asp Ile
# 590
- Met Asp Tyr Tyr Phe Ser Glu Lys Gly Phe Gl - #u Val Cys Arg Ile Asp
# 605
- Gly Ser Val Lys Leu Asp Glu Arg Arg Arg Gl - #n Ile Lys Asp Phe Ser
# 620
- Asp Glu Lys Ser Ser Cys Ser Ile Phe Leu Le - #u Ser Thr Arg Ala Gly
625 6 - #30 6 - #35 6 -
#40
- Gly Leu Gly Ile Asn Leu Thr Ala Ala Asp Th - #r Cys Ile Leu Tyr Asp
# 655
- Ser Asp Trp Asn Pro Gln Met Asp Leu Gln Al - #a Met Asp Arg Cys His
# 670
- Arg Ile Gly Gln Thr Lys Pro Val His Val Ty - #r Arg Leu Ser Thr Ala
# 685
- Gln Ser Ile Glu Thr Arg Val Leu Lys Arg Al - #a Tyr Ser Lys Leu Lys
# 700
- Leu Glu His Val Val Ile Gly Gln Gly Gln Ph - #e His Gln Glu Arg Ala
705 7 - #10 7 - #15 7 -
#20
- Lys Ser Ser Thr Pro Leu Glu Glu Glu Asp Il - #e Leu Ala Leu Leu Lys
# 735
- Glu Asp Glu Thr Ala Glu Asp Lys Leu Ile Gl - #n Thr Asp Ile Ser Asp
# 750
- Ala Asp Leu Asp Arg Leu Leu Asp Arg Ser As - #p Leu Thr Ile Thr Ala
# 765
- Pro Gly Glu Thr Gln Ala Ala Glu Ala Phe Pr - #o Val Lys Gly Pro Gly
# 780
- Trp Glu Val Val Leu Pro Ser Ser Gly Gly Me - #t Leu Ser Ser Leu Asn
785 7 - #90 7 - #95 8 -
#00
- Ser
- <210> SEQ ID NO 3
<211> LENGTH: 5000
<212> TYPE: DNA
<213> ORGANISM: Arabidopsis thaliana
- <400> SEQUENCE: 3
- tgtcgaagtt tccatggaag attgtgacca cgacgatgaa gctgaagatt ct - #ggtcacgt
60
- tgaaaacctt tgttacagat ttcgcaaacg aatcgattcg ttgccataag tg - #ttttaggt
120
- gacaaagcta tcacttcagc gtctggatct gaatttagac aatcagtgag aa - #caactaaa
180
- aacagaaaat ttcaaactca aaaaacagaa aaaaaaaagt ttggattttt ga - #gaagtacc
240
- aggcattcca ggaagattcc gtttcttctt cccgacggat ttaggagtta ga - #ttttggtt
300
- tccggtcgat gagacgcttg catcgccgga aactgtagag gaattatcta aa - #tcaaccgg
360
- catgtttcaa agatactaaa ttccaatctt tgaacacaaa aaggaagaag ca - #aatctcag
420
- ctcagctcaa tctagggttt atcatcctcc tcctactctg tttagtctct ct - #ttctctct
480
- ctcttcttca gctaccagtc aatctgcttt tcgtaaaaat ctccttttcc cc - #tttccgcc
540
- accaaacttt tctgataact cactctctga cctctcttct tcaaaaagat tt - #aaaacccc
600
- caaaagaaaa agaaaaaaaa tcaaaacttc attacccaag aaatctctta at - #catttaac
660
- ccagactctt tcttctccac acgcatcttt tatccaccgt ccaccgatct ga - #tccaacgg
720
- ctgagatttc accggagacg agttatcctt actacttccg gcttgtttct ct - #ctgaagaa
780
- tcaccggaaa aaaaaataag gcggcttgtg tgtgagactt tgtgtgaaag ct - #tcaacctt
840
- ttttttcttt ttctttggct tgtccaagaa aaaggagcct tcttcttctt tt - #ctctctct
900
- ggagacaatt atactaattt ttttcttttc aacttttcac cctttttttt tt - #gttaacaa
960
- acatttttta tacataattg tgtcgacttt caagttccaa gtatctaaat ct - #gtattttg
1020
- gactcccatg caaataatta aaatagaata atctttttgt agattttaaa tt - #gaaaacgg
1080
- tgtagaaagg ttaaaagcac caaacaaaac gagtaaatag atattgtaat aa - #ttttttca
1140
- cctttatgga aaagattata tcatagacga tgtacacaga tgaaaattag aa - #aatggcat
1200
- gtgaatatat gcagtaccca atgaatgcaa tatcaggttt gtattatttt tc - #tattgtat
1260
- ctctacatgt tacgtaatca aacgatcaag taatttatta atattgtcga tg - #gcgtagaa
1320
- attataaatt tattttatgt cattgtttac tatatagatt ttgagctaaa cg - #acttattt
1380
- tgtcaaaaga tatatccgtg tttggtttaa gattgggttt tagtatttcc aa - #tattaatc
1440
- taaattctta gcttatgaac atgtcaataa acaaaaaaat tattttactg tc - #actgtcct
1500
- tagacgggga caaaggaggg tattaccgtc gcgttgtcgg accgtaaaat aa - #ttaaccaa
1560
- attttgttgt tgaacgaata acatttttta ctgtgggaat ttgtcgtgta gc - #attacgtt
1620
- cgaaatcgca atttgttttc ttctttgtgg gtgtatattt ctggttaacg aa - #actataac
1680
- ccaatttaat gcaatgttcg tctgtttttg ttgactttga cccttttttg gt - #aatattcg
1740
- ttcagctttt gttttaacgt tttcattgcc ttgtaggcat ctgagaagct ca - #gattctga
1800
- cacgtgtctt ttgttatctg aatttgcatc cgttggataa acatgacgct ga - #caggtgga
1860
- ttgaaaagta accagcttgg atttctgtgt atatgttaca ccgccacttc cc - #ttaatttc
1920
- ttcgttctta gttaaaataa aaaaggttta atttatgagt aaaagtatgt aa - #aacgacaa
1980
- cgattactat aagaattaaa atttatcttt gcttagtaat ttgcacttaa ga - #ttggattc
2040
- aaattttgta aaaagcgaat gttacatata tgtccattga aaaaattgca tt - #tgacttta
2100
- caagcattga aattaattaa tttgggaccc ctttttttgt tagtttcaaa gg - #aagaatta
2160
- ttttaggctg agatgggtcc ctccataaac tcactattct gccagcatac aa - #attcctta
2220
- acatatggtc caaatagcag ttccaaccac tagtatccaa taataatctg aa - #caaattat
2280
- ctttcttttt ttttcctgat aatcttgtat ttgtttgttc aatgagctta at - #acgtatat
2340
- tagttatgac ttataactaa atactttgac tcacttgatc cgtacacatt ga - #tttcgttt
2400
- attcaaatcc gaacaacgta atgatctttt tgggccgagt tatttgtatt ct - #caacctga
2460
- gtccaaccat gctttatggg cttttctgtt tatttatgca tgtaaagttt at - #aatgcttg
2520
- caaataacca catattgtat gaatgtaatt actatgattt aagggcactg ct - #tttctgtt
2580
- ttcacgttgt tttcgaaatt gctattgcgt gtgatatctg tgttggacca at - #tattgaaa
2640
- aggacaaggc tgactctggt ttttaatgag tagtccccat gggagttatg tt - #catttacc
2700
- acacattttt ttgtatagta tagtatgagt ttttatttga tatcttttat ct - #tcggaaaa
2760
- taaatggttc aaattgtttg tctaaaaatg cacacatgaa tatcttgtgg tc - #tcacacaa
2820
- ttgtaggaaa caaattaata tttgttgcga aaataatgtt attattttat ca - #tacgaaat
2880
- cctagagaaa atggtggcaa aagaggcaaa gactaaacta atgaatttaa aa - #tatgaaaa
2940
- tgatggaatg actggtttac caatattaca gtatattgta attttataaa aa - #cgaatcct
3000
- gaagaagagg gcaaacccca agaccacgca aatcagtcta caaatatgaa aa - #tttccaat
3060
- aactagaaaa acatgtgcat ttatcttttt ccatcattcg gatttttaca at - #ggaaattt
3120
- tgaccactga gcgcaagtgt tatagtattt tattattatc caatattaat at - #cattattc
3180
- ggatccatgc attctatata actatgtcca ccatcttact tgtgtctatg tt - #gcaacttc
3240
- aacgtcgtat atatataggg attgttgtca cgaatacaat gctaattaag ga - #agattgtg
3300
- acttctcgga aaatttagaa ctaattaaga gtggaactaa aatgccaatg aa - #aatagcct
3360
- aaatcaaagg agaaccacaa atataaattg gaagacctta aaaaacaatt aa - #acgaggac
3420
- gaaacaaatt ttggaatcat caattatacg aaaaaaagaa gaaagaaaaa ag - #aggtttca
3480
- tgaatcacag tagtgctgac aatcttcgaa ccatttgtgg gtttcataca at - #cgatcacc
3540
- aatagaacaa aagagaaaca gaggaacaga aagaatagaa ggagtgggaa gt - #gtatgagg
3600
- aagctgtgtc cgaacataga caaagacgat ggtctggaga cggtgttgga ag - #ttccgata
3660
- ccggaggaga tgttttccgg tatgggcaac aacgttgcac ttaggtggca aa - #atatgatg
3720
- acgtggatga aagctcaaac gtctgataaa tggtcgcaac cgcttatcgc cg - #ctcgtatc
3780
- aacgagctcc ggttccttct ctacctcgtt ggctcgcctc ttatacctct cc - #aggttcaa
3840
- gtcggtcact ctgttcataa gcccgtcaaa gattgctcca ttgtaagtca tt - #caaaatca
3900
- atccttatga aaacataaca aagatgttga aaatatgatt cctctttttt tt - #ttcttttt
3960
- ttcttttatg atcaaaaccc aaaaaagtca ttaccctgct tcgtaagtat tc - #aacataaa
4020
- gttgttaatc catgtgttgt actctgcaag tctgcattac attattcatc gt - #acacagag
4080
- tcatcaactt cagtttcatt gtttttttgc ttatgaatta cgattgcagc aa - #gcttcaac
4140
- ggcgaaatac attgtacagc agtacatagc agcgacggga ggaccacagg cg - #ttaaacgc
4200
- cgtgaacagc atgtgcgtca cgggacaagt gaagatgacg gcgtcggagt tt - #catcaagg
4260
- agatgattcg ggcgttaatc taaagagcaa cgacgaaatg ggtggtttcg tt - #ttatggca
4320
- aaaggatcca gatctttggt gtttggagct cgtcgtctcc ggttgcaaag tg - #gatatgtg
4380
- gaagcaacgg tcggctttca tggcgacatt cctctaacca gcaaactccg gc - #gtctacsg
4440
- gaacgccaar acctctccgc cggtttwtac aggtccaatc cggttattga tt - #tttttttk
4500
- gatgtaatgt ccggttctca aaatgttgaa ccggtggttt atttattgtt tg - #gagcaggg
4560
- gttaratcct cgttcgacgg cgaatctgtt tcttgacgca aacgtgtatc gg - #agagaaga
4620
- taatcaacgg tgaggattgc tttatcttga aactggagac gagtccggcg gt - #tcgagaag
4680
- ctcaaagcgg tccgaatttt gagataattc atcacacgat atggggttat tt - #tagtcaaa
4740
- gatcgggact tttgattcag ttcgaagatt cgcggctttt gagaatgagg ac - #caaggaag
4800
- acgaagatgt cttctgggag actagtgctg agtcggtgat ggatgattac cg - #atacgttg
4860
- acaatgtgaa catcgctcac ggcgggaaaa catcggtcac ggttttccgg ta - #cggtgaag
4920
- cgtcggcgaa tcatcggaga cagatgacgg agaagtggag gatagaagaa gt - #tgatttta
4980
# 500 - #0
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